november9san diego poster correctio october 26

HIV induces complement C3 in primary human astrocytes indirectly through a NFkB-dependent pathwayJadwiga Nitkiewicz, Wei Chao, Seon-Young Kim, Luni Nemdad, Devanand Sarkar, Paul Fisher and David J. VolskyMolecular Virology Division, St. Lukes-Roosevelt Hospital Center, and Department of Pathology Columbia University Medical Center, New York, NY 10019INTRODUCTIONInnate immunity is believed to play an important role in protecting brain against HIV infection and development of HIV-associated dementia (HAD). Complement C3 is a key component of the innate immunity system but in models of other neurodegenerative diseases such as multiple sclerosis, uncontrolled C3 production can lead to tissue injury and inflammation. C3 is secreted by several cell types in the brain including astrocytes. We and others have previously shown that HIV interaction with astrocytes in vitro alters cellular gene expression, induces interleukin-6, and disrupts important physiological functions of the cells such as glutamate transport. Here we investigated the mechanism of C3 induction in human fetal astrocytes (HFA) exposed to HIV in vitro. Using transient transfection assays with a C3 promoter-driven indicator gene, real-time PCR, and C3 ELISA we found that exposure of astrocytes to HIV increased C3 promoter activity, mRNA expression, and protein production, indicating that HIV acts by transcriptional activation of the C3 gene. Studies with pharmacological inhibitors revealed that activation of C3 by HIV requires, among others, the NFkB and PTK signaling pathways; however, surprisingly, the C3 core promoter lacks NFkB and SP binding sites. Activation of the C3 promoter by HIV was absolutely dependent on the presence of the interleukin 1-beta/interleukin 6 responsive element at -109 to -90 and its sub-domain -106 to -100. The requirement for functional NFkB for HIV effect on C3 was confirmed by overexpresion of the NFkB inhibitor IkB-a in astrocytes; adenovirus-mediated transduction of IkB-a into HFA, but not of control virus, blocked nuclear translocation of NFkB and prevented induction of C3 promoter and mRNA. FIGURE 2A, BEFFECT OF PHARMACOLOGICAL INHIBITORS OF SIGNAL TRANSDUCTION PATHWAY ON C3 PROMOTER INDUCTION BY HIV IN ASTROCYTESFIGURE 2C EFFECT OF INHIBITORS OF NFKAPPA B (CAPE) and PTK (GENISTEIN) ON C3 GENE EXPRESSION AFTER HIV EXPOSURE OF PRIMARY ASTROCYTESFIGURE 1 A. DETECTION OF C3 PROTEIN SECRETION IN CELL SUPERNATANTS B. C3 GENE EXPRESSION IN EARLY (24H), AND LATE (7 D) TIMEPOINT TESTED IN CELL EXTRACTC. INDUCTION OF C3 PROMOTER AFTER 48H; TESTED IN CELL EXTRACTFIGURE 3A DETECTION OF p50 SUBUNITS OF ACTIVATED NFkappaBIN NUCLEAR EXTRACT (AFTER 30 MIN OF THE HIV EXPOSURE OF PRIMARY ASTROCYTES BY TRANSBINDING TM NFkappaB ASSAY KIT)

RPN800 TNF IL-1 HIV mock medium RPN 756 (0.5, 1pgP24/cell) FIGURE 3BDETECTION OF PYK-2 PROTEIN TYROSINE KINASE IN CELL EXTRACT (BY W-B AFTER 30 MIN OF HIV EXPOSURE OF HFA)ASTROCYTES INFECTED WITH ADENOVIRUS MUTANT MT32IkB (2HR PRIOR HIV EXPOSURE) FIGURE 5 A, B, CCell-mediumCell-MockCell-IL1Cell-HIVAd.vec-mediumAd.vec-IL1Ad.vec-HIVAd.vec-MockAd.IkBa-mt32-mediumAd.IkBa-mt32 -MockAd.IkBa-mt32 -HIVAd.IkBa-mt32 -IL1IMMUNOSTAINING FOR DETECTION OF p65 SUBUNITS ACTIVATED NFkappaB(images from Confocal Laser Scanning Microscope LSM multiphoton 510)

LUCLUCLUCLUCLUCLUCLUCXXXXXFIGURE 4A THE DELETION ANALYSIS OF C3 PROMOTER REGION IN HIV RESPONSE OF PRIMARY ASTROCYTES FIGURE 4BTHE MUTATION ANALYSIS OF C3 PROMOTER IN HIV RESPONSE 1. mutation C1 109 -(-90) IL-1/IL-6 2. mutation C2 106- (-100) IL6 3. mutation C3 116 (-100) IFN- gamma 4. mutation C4 181- (-175) IL6 5. mutation C5 615 (-604) NFkB 6. mutation C6 750 (-740) NFkBCONCLUSIONSEXPOSURE OF PRIMARY ASTROCYTES TO HIV

INDUCES ACTIVATION of COMPLEMENT C3 PROMOTER, INCREASED C3 mRNA TRANSCRIPTION, and SECRETION of C3 PROTEIN

INDUCTION of C3 GENE requires primarily PROLINE RICH PROTEIN TYROSINE KINASE PYK-2 , MAPK signaling pathways, and is NFkappaB dependent; through indirect mechanism- NFkB binding sites in C3 promoter are dispensable for promoter activity

CRITICAL RESPONSIVE ELEMENT on C3 PROMOTER in HIV-1 RESPONSE is LOCATED; between NUCLEOTIDES [-109- (90)] with SUBDOMAIN at [ 106- (-100]; and it includes IL1-, and IL-6 responsive elementsXINDUCTION OF C3 PROMOTER TESTED IN CELL EXTRACTDETECTION OF p50 SUBUNITS OF ACTIVATED NFkappaB HETERODIMER IN NUCLEAR EXTRACT (30MIN/HIV)FIG 1AFIG 1BFIG 1CFIG 2AFIG 2BFIG 2CFIG 5AFIG 5BFIG 5C

Chart1

1.5333333330.251661148

1.8750.275

3.190.69

0.8750.075

1.010.01

48h

C3 Promoter Activity (RLU, FC)

inhibitors 2b graph

317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract

Relative Luciferase Activity [RLU]

graph cont inhib 2b

1577.9917269723

62.52

113.6055512755

150.2385979883

48h

cell extract


kontrole do inhib 2b

HIVmedmock cIL-1TNF-mediummock c

average31111515762.51115

25.51316156.5551316

40.011118158.170.531118

26.95911156.461.97911

average30.82111515762.51115

SD7.991726972323.60555127550.95393920147.778553850223.6055512755

Ttest0.03534436910.00000031680.0006591335

IL-1 betaTNF-alfamediummock

15762.51115

156.5551316

158.170.531118

156.461.97911

av15762.51115

SD0.95393920147.778553850223.6055512755

0.0006591335

inhibitors2b

HIVIL-1 betaTNF- alfamediummockCape/HIVCape/mediumPDTC/HIVPDTC/mediumAG18/HIVAG18/mediumU126/HIVU126/mediumWortmanin/HIVWortmanin/mediumSQ-22536/HIVSQ-22536/mediumCalphostin/HIVCalphostin/medium

3115762.5111533.61311.51111.43733382137.6302825

7.99172697230.95393920147.778553850223.60555127550.23859798830.35694257240.16165807540.50.88573886292.11660104890.71103650870.41508031355.36055967230.86694867214.89672339434.43030472996.55743852432.6457513111

19-Jul

HIVmediummockCape/HIVCape/mediumPDTC/HIVPDTC/mediumAG18/HIVAG18/mediumU126/HIVU126/mediumCalphostin/HIVCalphostin/medium

av31111533.61311.51111.437332825

SD7.991726972323.60555127550.23859798830.35694257240.16165807540.50.88573886292.11660104890.71103650870.41508031356.55743852432.6457513111

controle do graph inhib2b wl


1570.9539392014

62.57.7785538502

112

153.6055512755

48h

cell extract


wl graph 1 Elisa p50NFkB

317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract


graphknock 5000vpC3 prom

317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract


wlgraph knockout Il1 Il6 5000vp

1570.9539392014

62.57.7785538502

112

153.6055512755

48h

cell extract


knockout Il1, Il6 5000vp

317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract


knockout5000Il1,Il6

1570.9539392014

62.57.7785538502

112

153.6055512755

48h

cell extract


wl graph ad 5000 C3 prom bz Il1

317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract


daneknockout 5000vp promotor C3

1570.9539392014

62.57.7785538502

112

153.6055512755

48h

cell extract


1 EL p50NFkB

317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract


EL p50 controls

1570.9539392014

62.57.7785538502

112

153.6055512755

48h

cell extract


mC3RNA 24h, 3d signal transd


0.280.0115470054

0.370.0251661148

0.40.0057735027

0.530.0057735027

0.0260.0005773503

0.0240.0005773503

0.5450.0025166115

-0.010.0005773503

30min

nuclear extract

OD value [450nm]

inhib2A wlasc grapg bez Il1

0.280.0115470054

0.370.0251661148

0.40.0057735027

0.530.0057735027

0.0260.0005773503

0.0240.0005773503

0.5450.0025166115

-0.010.0005773503

30min

nuclear extract

OD value [450nm]

graph Inhib 2A bez Il1

471.5275252317

242.5166114784

221.443375673

4.90.1527525232

6.20.2645751311

4.250.2306512519

38.31.5275252317

7.10.3511884584

6.90.2

48h

cell extract


inhibitors 2A

inhibitors 2A

1312

451.5275252317

222.5166114784

20.61.443375673

9.90.1

5.30.1527525232

6.20.2645751311

4.180.2306512519

124.31.5275252317

461

6.80.3511884584

6.30.2

48h

cell extract


grph C3 protein1

1312

451.5275252317

222.5166114784

20.61.443375673

9.90.1

5.30.1527525232

6.20.2645751311

4.180.2306512519

124.31.5275252317

461

6.80.3511884584

6.30.2

48h

cell extract


daneC3 proteina

1312

451.5275252317

222.5166114784

20.61.443375673

9.90.1

5.30.1527525232

6.20.2645751311

4.180.2306512519

124.31.5275252317

461

6.80.3511884584

6.30.2

48h

cell extract


Q-PCR 24, 7ddane

Exp 02/05IL-1 beta 1ng/ml][20ng/ml]Ad-IkB/IL-1Ad-IkB/IL-6Ad-IkB/mockAdenovirusAd/mockAd/IL-1

Br52/2

systemIL-1 betaIL-6mockmediumAd-IkB/IL-1Ad-IkB/IL-6Ad-IkB/mockAdenovirus-IkB/mediumAd/IL-1Ad/IL-6Ad/mockAdenovirus/medium

average131452220.69.95.36.24.18124.3466.86.3

11314422219.75.56.63.94125457.26.5

2129471918.59.55.36.14.4126476.86.3

31334624219.65.26.54.2123466.56.1

average13145.666666666722.2520.6259.65.33333333336.44.18124.6666666667466.83333333336.3

SD21.52752523172.51661147841.4433756730.10.15275252320.26457513110.23065125191.527525231710.35118845840.2

Ttest

Ad-IkB/mockAdenovirus-IkBAdenovirusAd/mockAd/IL-1Ad/IL-6

6.97.1124.346

Ad-IkB/mockAdenovirus-IkB/medium6.57.212545

6.24.256.36.812647

6.63.946.16.512346

6.14.46.36.8333333333124.666666666746

6.54.20.20.35118845841.52752523171

6.44.18

0.26457513110.2306512519

Q-PCR 24, 7ddane

1262

451.5275252317

242.5166114784

221.443375673

9.90.1

5.70.1527525232

4.250.2306512519

6.20.2645751311

6.90.2

7.10.3511884584

124.31.5275252317

461

5000vp/cell

cell extract


wykresy Q-PCR mRNA24, 7d

wykresy Q-PCR mRNA24, 7d

471.5275252317

242.5166114784

221.443375673

4.90.1527525232

6.20.2645751311

4.250.2306512519

38.31.5275252317

7.10.3511884584

6.90.2

48h

cell extract


wl wykr C3 promoter

471.5275252317

242.5166114784

221.443375673

4.90.1527525232

6.20.2645751311

4.250.2306512519

38.31.5275252317

7.10.3511884584

6.90.2

48h

cell extract


wykr promoter C3 ind

Exp 02/05Ad 5000vp/cellAd-IkB/mockAd-IkB/HIV

Br52/2Adenovirus-IkB

systemHIV+mockmediumAdenovirus-IkBAd-IkB/mockAd-IkB/HIVAdenovirusAd/mockAd/HIV+

average4724224.256.24.96.97.138.3

14822213.946.64.76.57.236

2491918.54.46.14.96.36.837

34624214.26.54.66.16.539

average47.522.2520.6254.186.44.73333333336.36.833333333337.3333333333

SD1.52752523172.51661147841.4433756730.23065125190.26457513110.15275252320.20.35118845841.5275252317

Br52/2

systemHIV+mockmediumAd-IkB/HIV+Ad-IkB/mockAd-IkB/mediumAd/HIV+Ad/mockAd-medium

average4724224.96.24.2538.37.16.9

14822214.76.63.94367.26.5

2491918.54.96.14.4376.86.3

34624214.66.54.2396.56.1

average47.522.2520.6254.73333333336.44.1837.33333333336.83333333336.3

SD1.52752523172.51661147841.4433756730.15275252320.26457513110.23065125191.52752523170.35118845840.2

dane i wykr promoter C3

0.280.0115470054

0.370.0251661148

0.40.0057735027

0.530.0057735027

0.0260.0005773503

0.0240.0005773503

0.5450.0025166115

-0.010.0005773503

30min

nuclear extract

OD value [450nm]

Elisa NFkB controls z knockout

systemcontrol+Competition cIL-1 betaIL-6HIV+mockmedium

average0.2170.0330.2040.1240.1560.0620.047

10.2170.2170.1240.160.060.047

20.2140.0360.2080.1210.150.0610.044

30.220.0330.2040.1250.1620.0640.049

average0.2170.03450.20966666670.12333333330.15733333330.06166666670.0466666667

SD0.0030.00212132030.00665832810.0020816660.00642910050.0020816660.0025166115

HIV [2pg]HIV [4pg]TNF[10U/ml]IL-1[5ng/ml]control extmediumpositive controlcompetition control

av0.20.30.350.530.0270.0240.545-0.01

0.30.40.40.530.0260.0230.54-0.01

0.280.350.390.520.0250.0240.542-0.011

av0.260.350.380.52666666670.0260.02366666670.5423333333-0.0103333333

SD0.05291502620.050.02645751310.00577350270.0010.00057735030.00251661150.0005773503

Ttest0.00156259920.00035917490.00002060.00000000.00000000.0000007

HIV [1pg]HIV [2.5pg]TNF-alfaIL-1 betamockmediumpositive controlcompetition control

av0.280.370.40.530.0260.0240.545-0.01

0.30.40.40.530.0260.0230.54-0.01

0.280.350.390.520.0250.0240.542-0.011

av0.28666666670.37333333330.39666666670.52666666670.02566666670.02366666670.5423333333-0.0103333333

SD0.01154700540.02516611480.00577350270.00577350270.00057735030.00057735030.00251661150.0005773503

Ttest0.00000255570.00001811070.00000000.00000000.00000000.0000002

wykres Elisa NFkB contr z Knock

cell signalling involved in the activation of C3 gene expression

HIVmockmediumCape/HIVCape/mockCape/mediumGenistein/HIVGenistein/mockGenistein/mediumAG17/HIVAG17/mockAG17/medium

24h1410.672.90.580.4931.170.96.30.630.69

13.70.90.592.40.550.512.61.280.85.80.90.77

15.21.10.712.20.610.522.91.41.15.70.860.82

av14.310.65666666672.50.580.50666666672.83333333331.28333333330.93333333335.93333333330.79666666670.76

SD0.79372539330.10.06110100930.36055512750.030.01527525230.20816659990.11503622620.15275252320.32145502540.145716620.0655743852

test

3d15.540.6560.783.50.40.93.12.11.86.70.780.5

16.40.70.793.40.520.913.22.21.66.40.980.6

16.30.630.822.950.490.893.52.31.56.20.890.67

av16.080.6620.79666666673.28333333330.470.93.26666666672.21.63333333336.43333333330.88333333330.59

SD0.47031904070.0353836120.020816660.29297326390.062449980.010.20816659990.10.15275252320.25166114780.1001665280.0854400375

SD


24h14.310.65666666672.50.580.50666666672.83333333331.28333333330.93333333335.93333333330.79666666670.76

3d16.080.6620.79666666673.28333333330.470.93.26666666672.21.63333333336.43333333330.88333333330.59

SD240.79372539330.10.06110100930.36055512750.030.01527525230.20816659990.11503622620.15275252320.32145502540.145716620.0655743852

SD3d0.47031904070.0353836120.020816660.29297326390.062449980.010.20816659990.10.15275252320.25166114780.1001665280.0854400375

wykres Elisa NFkB contr z Knock

14.316.08350.79372539330.47031904071

10.6620.93333333330.10.0353836120.1527525232

0.65666666670.79666666670.96333333330.06110100930.020816660.118462371

2.53.28333333333.33333333330.36055512750.29297326390.4509249753

0.580.470.75666666670.030.062449980.0585946528

0.50666666670.90.80.01527525230.010.1

2.83333333333.26666666674.10.20816659990.20816659990.1732050808

1.28333333332.22.46666666670.11503622620.10.2516611478

0.93333333331.63333333332.09333333330.15275252320.15275252320.1101514109

5.93333333336.433333333311.40.32145502540.25166114781.1532562595

0.79666666670.88333333330.91333333330.145716620.1001665282.74

0.760.590.13233333330.06557438520.08544003750.0924463809

24h

3d

7d

cell extract

Relative quantification; Fold change

Cell signalling involved in the activation of C3 gene expression

OD dane NFkB

000.79372539330.4703190407

000.10.035383612

000.06110100930.02081666

000.36055512750.2929732639

000.030.06244998

000.01527525230.01

000.20816659990.2081665999

000.11503622620.1

000.15275252320.1527525232

000.32145502540.2516611478

000.145716620.100166528

000.06557438520.0854400375

24h

3d

cell extract

Fold change [C3 mRNA]

neutralization of Il6

neutralization of Il6

135.33333333336.1101009266

654.3588989435

66.33333333331.5275252317

5.56666666670.5131601439

5.50.5

5.53333333330.9237604307

100.333333333317.616280349

1118.1853527719

85.33333333338.9628864398

33.33333333334.9328828623

37.66666666678.5049005481

33.333333333310.4083299973

214.3588989435

21.66666666678.9628864398

21.66666666678.0829037687

53.66666666675.5075705473

51.66666666676.6583281185

495.2915026221

65.666666666711.0151410946

47.33333333332.5166114784

492.6457513111

3.66666666670.5773502692

5.33333333330.5773502692

3.66666666670.5773502692

48h

cell extract

Re;ative Luciferase Activity [RLU]

Inhib December grap1 July 20,07

135.33333333336.1101009266

654.3588989435

66.33333333331.5275252317

5.56666666670.5131601439

5.50.5

5.53333333330.9237604307

100.333333333317.616280349

1118.1853527719

85.33333333338.9628864398

33.33333333334.9328828623

37.66666666678.5049005481

33.333333333310.4083299973

214.3588989435

21.66666666678.9628864398

21.66666666678.0829037687

53.66666666675.5075705473

51.66666666676.6583281185

495.2915026221

65.666666666711.0151410946

47.33333333332.5166114784

492.6457513111

3.66666666670.5773502692

5.33333333330.5773502692

3.66666666670.5773502692

48h

cell extract


GrapInhDec July 20,07

HIVmockmediumCape/HIVCape/mockCape/mediumP38/HIVP38/mockP38/mediumSB580/HIVSB580/mockSB580/mediumSB190/HIVSB190/mockSB580/mediumAG17/HIVAG17/mockAG17/mediumJNK/HIVJNK/mockJNK/mediumGenistein/HIVGenistein/mockGenistein/medium

135.33333333336566.33333333335.56666666675.55.5333333333100.333333333311185.333333333333.333333333337.666666666733.33333333332121.666666666721.666666666753.666666666751.66666666674965.666666666747.3333333333493.66666666675.33333333333.6666666667Average

6.11010092664.35889894351.52752523170.51316014390.50.923760430717.6162803498.18535277198.96288643984.93288286238.504900548110.40832999734.35889894358.96288643988.08290376875.50757054736.65832811855.291502622111.01514109462.51661147842.64575131110.57735026920.57735026920.5773502692SD

0.00008431090.00001958870.00001947480.00002075310.02798383280.00100840240.024151360.00113383070.00774020590.00827556490.00024601170.00122031550.04907193280.01557401090.00690512192.51661147840.0055620.00001740970.00001942550.0000174097Ttest

GrapInhDec July 20,07

135.33333333336.1101009266

37916.8226038413

654.3588989435

66.33333333331.5275252317

5.56666666670.5131601439

5.50.5

5.53333333330.9237604307

100.333333333317.616280349

1118.1853527719

85.33333333338.9628864398

33.33333333334.9328828623

37.66666666678.5049005481

33.333333333310.4083299973

214.3588989435

21.66666666678.9628864398

21.66666666678.0829037687

53.66666666675.5075705473

51.66666666676.6583281185

495.2915026221

65.666666666711.0151410946

47.33333333332.5166114784

492.6457513111

3.66666666670.5773502692

5.33333333330.5773502692

3.66666666670.5773502692

48h

cell extract


Inhibitors December or1 graph

1NFkB ELISA

PROTEINAVERAGESD

IL-1141.66666677.637626158

TNF-alfa108.66666673.214550254

HIV [0.5]10.333333331.040833

HIV [1pg]19.166666672.020725942

HIV [2.5pg]20.766666671.078579312

mock0.30.2

medium0.2433333330.049328829

IL-1512.66666676.429100507

TNF-alfa457.76666678.494900431

HIV [0.5]87.333333333.214550254

HIV [1pg]191.66333336.345079458

HIV [2.5pg]236.666666712.58305739

mock3.510.5

medium30.1

IL-1 betaTNF-alfaHIV [0.5pg]HIV [1pg]HIV [2.5pg]mockmedium

av3d141.66108.6610.3319.1620.760.30.25

av7d512.66457.7687.33191.66236.661412.5

SD7.63.21.0421.0732

Sd6.48.43.26.312.532.5

IL-1HIV [0.5pg]HIV [1pg]HIV [2.5 pg]mock cmediumIL-1HIV [0.5]HIV [1pg]HIV [2.5 pg]mock cmedium

average10.33333333333.24.85.33333333331.066666666718.76666666673.53333333334.26666666673.73333333331.23333333331.1666666667

SD0.57735026920.11.31148770490.28867513460.20816659990.36055512750.49328828620.35118845840.30550504630.46188021540.11547005380.1154700538

Ttest0.0000127030.00008921650.00822227110.00003178290.00001350040.0004205250.00008733320.0008104185

IL-1 betaHIV [0.5pg]HIV [1pg]HIV [2.5pg]mockmedium

av24h13.333.24.85.5511.1

av7d93.64.36.51.21.6

SD0.570.11.30.20.20.4

SD0.50.350.30.40.20.1

HIV [0.5pg]HIV [1pg]HIV [2.5pg]mockmedium

24h3.24.85.5511.1

7d3.64.36.51.21.6

SD0.11.30.20.20.4

SD0.350.30.40.20.1

10.33333333330.5773502692

3.20.1

4.81.3114877049

5.33333333330.2886751346

1.06666666670.2081665999

10.3605551275

8.76666666670.4932882862

3.53333333330.3511884584

4.26666666670.3055050463

3.73333333330.4618802154

1.23333333330.1154700538

1.16666666670.1154700538

48h

cell extract

Fold change [Q-PCR]

000.570.5

000.10.35

001.30.3

000.20.4

000.20.2

000.40.1

24h

7d

cell extract

C3 mRNA (FC)

000.10.35

001.30.3

000.20.4

000.20.2

000.40.1

24h

7d

mRNA (Fold Change)

13.3390.570.5

3.23.60.10.35

4.84.31.30.3

5.556.50.20.4

11.20.20.2

1.11.60.40.1

24h

7d

cell extract

Fold hange [mRNA]

13.3390.570.5

3.23.60.10.35

4.84.31.30.3

5.556.50.20.4

11.20.20.2

1.11.60.40.1

24h

7d

cell extract

Fold hange [mRNA]

9.750.75

6.30.7

1.5333333330.251661148

1.8750.275

3.190.69

0.8750.075

1.010.01

48h

cell extract

Fold change [RLU]

9.750.75

6.30.7

1.5333333330.251661148

1.8750.275

3.190.69

0.8750.075

1.010.01

48h

cell extract

Fold change [RLU]

9.750.75

6.30.7

1.5333333330.251661148

1.8750.275

3.190.69

0.8750.075

1.010.01

48h

cell extract

Fold change [RLU]

9.750.75

6.30.7

1.5333333330.251661148

1.8750.275

3.190.69

0.8750.075

1.010.01

48h

cell extract

Fold change [RLU]

IL-1betaTNF-alfaHIV [0.5pg]HIV [1pg]HIV [2.5pg]mockmedium

9.756.31.5333333331.8753.190.8751.01

SD0.750.70.2516611480.2750.690.0750.01

00.251661148

00.275

00.69

00.075

00.01

48h

Relative Luciferase Activity [RLU] Fold Change

0.2170.003

0.0330.0021213203

0.2040.0066583281

0.1560.0064291005

0.0620.002081666

0.0470.0025166115

0.0560.0026457513

0.060.001

0.040.01

nuclear extract

OD value [450nm]

0.2170.003

0.0330.0021213203

0.2040.0066583281

0.1560.0064291005

0.0620.002081666

0.0470.0025166115

0.0560.0026457513

0.060.001

0.040.01

nuclear extract

OD value [450nm]

0.2170.003

0.0330.0021213203

0.2040.0066583281

0.1560.0064291005

0.0620.002081666

0.0470.0025166115

0.0560.0026457513

0.060.001

0.040.01

nuclear extract

OD value [450nm]

controls

osobno IL-1, IL-6

systemcontrol+Competition cIL-1 betaHIV+mockmediumCape/HIV+Cape/mockCape/medium

average0.2170.0330.2040.1560.0620.0470.0560.060.04

10.2170.2170.160.060.0470.0540.060.03

20.2140.0360.2080.150.0610.0440.0550.0590.05

30.220.0330.2040.1620.0640.0490.0590.0610.04

average0.2170.03450.20966666670.15733333330.06166666670.04666666670.0560.060.04

SD0.0030.00212132030.00665832810.00642910050.0020816660.00251661150.00264575130.0010.01

wirusy

systemcontrol+Competition cmockmediumHIV+Ad-v/HIV+Ad-v/mockAd-vAd-IkB/HIV+Ad-IkB/mockAd-IkBCape/HIVCape/mock

average0.2170.0330.0620.0470.1560.1320.1130.1010.0510.0530.0490.0560.06

10.2170.060.0470.160.1340.1150.1080.0560.0530.050.0540.06

20.2140.0360.0610.0440.150.1380.1180.0990.0530.0560.0470.0550.059

30.220.0330.0640.0490.1620.1240.1060.0960.0440.050.050.0590.061

average0.2170.03450.06166666670.04666666670.15733333330.1320.1130.1010.0510.0530.0490.0560.06

SD0.0030.00212132030.0020816660.00251661150.00642910050.00721110260.0062449980.0062449980.0062449980.0030.00173205080.00264575130.001

0.2170.003

0.0330.0149899967

0.2040.0066583281

0.1240.002081666

0.1560.0357258077

0.1320.0072111026

0.1130.006244998

0.1010.006244998

0.0510.006244998

0.0530.003

0.0490.0017320508

0.0560.0026457513

0.060.001

0.0620.002081666

0.0470.0025166115

cell extract

OD value [450nm]

IL-6HIVmockmediummouse IgGMoAb anti-IL6mouse IgG+HIVMoAb anti-IL6+HIV

Fch1.81.510.91.41.041.71

SD

4.52.82.71.83.92.3

42.72.82.23.62.1

3.73.22.662.23.31.8

SD0.40.20.20.150.30.260.40.28

Fold Ch1.81.510.91.41.041.71

1.80.4

1.50.2

10.2

0.90.15

1.40.3

1.040.26

1.70.4

10.28

48h

cell extract

Fold change [RLU]

1.80.4

1.50.2

10.2

0.90.15

1.40.3

1.040.26

1.70.4

10.28

48h

cell extract

Fold change [RLU]

1570.9539392014

62.57.7785538502

317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.7

11.50.5

110.8857388629

11.42.1166010489

430.7110365087

300.4150803135

385.3605596723

210.8669486721

37.64.8967233943

304.4303047299

286.5574385243

252.6457513111

48h

cell extract


1570.9539392014

62.57.7785538502

317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.7

11.50.5

110.8857388629

11.42.1166010489

430.7110365087

300.4150803135

385.3605596723

210.8669486721

37.64.8967233943

304.4303047299

286.5574385243

252.6457513111

48h

cell extract


IL-1TNF-HIVmediummockCape/HIVCape/mediumPDTC/HIVPDTC/mediumAG18/HIVAG18/mediumU126/HIVU126/mediumWortmanin/HIVWortmanin/mediumSQ-22536/HIVSQ-22536/mediumCalphostin/HIVCalphostin/medium

15762.5average31111533.61311.51111.43733382137.6302825

156.55525.513163.1234.01213.091212.1936323320.4632.7834.462724

158.170.5340.0111182.7253.38413.0911.510.712.2353343.662242.5725.62223

156.461.9726.959113.1523.40412.811110.461336.732.637.3420.5437.4529.943528

15762.5average30.82111533.612.996666666711.511.086666666711.44330382137.6302825

0.95393920147.7785538502SD7.991726972323.60555127550.23859798830.35694257240.16165807540.50.88573886292.11660104890.71103650870.41508031355.36055967230.86694867214.89672339434.43030472996.55743852432.6457513111

0.00000320.0006591335Ttest0.03534436910.00452958730.00550742530.01506078120.02077571580.01074868790.21012594370.09889791720.83434125080.0035112510.04869066430.0029959170.01043022450.03959261940.0179479132

tosamo z Il1

IL-1 betaTNF- alfaHIVmediummockCape/HIVCape/mediumPDTC/HIVPDTC/mediumAG18/HIVAG18/mediumU126/HIVU126/mediumWortmanin/HIVWortmanin/mediumSQ-22536/HIVSQ-22536/mediumCalphostin/HIVCalphostin/medium

15762.531111533.61311.51111.44330382137.6302825

156.55525.513163.1234.01213.091212.1936323320.4632.7834.462724

158.170.5340.0111182.7253.38413.0911.510.712.2353343.662242.5725.62223

156.461.9726.959113.1523.40412.811110.461336.732.637.3420.5437.4529.943528

15762.530.82111533.612.996666666711.511.086666666711.44330382137.6302825

SD0.95393920147.77855385027.991726972323.60555127550.23859798830.35694257240.70.50.88573886292.11660104890.71103650870.41508031355.36055967230.86694867214.89672339434.43030472996.55743852432.6457513111

0.00000320.00065913350.03534436910.00452958730.00550742530.01506078120.02077571580.01074868790.21012594370.09889791720.83434125080.0035112510.04869066430.0029959170.01043022450.03959261940.0179479132


0.20.30.350.530.0270.0240.545-0.01

0.220.320.360.550.0230.0220.546-0.011

0.210.330.3670.60.0210.0230.546-0.012

av

SD

MBD0002EB81.xls

C3protein wykres or

141.66512.667.66.4

108.66457.73.28.4

10.3387.331.043.2

19.16191.6626.3

20.762371.0712.5

0.311.533

0.2410.5

C

3d

7d

supernatants

C3 Protein [ng/ml]

inhibitors 2b graph

317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract


graph cont inhib 2b

1577.9917269723

62.52

113.6055512755

150.2385979883

48h

cell extract




average31111515762.51115

25.51316156.5551316

40.011118158.170.531118

26.95911156.461.97911

average30.82111515762.51115

SD7.991726972323.60555127550.95393920147.778553850223.6055512755

Ttest0.03534436910.00000031680.0006591335


15762.51115

156.5551316

158.170.531118

156.461.97911

av15762.51115

SD0.95393920147.778553850223.6055512755

0.0006591335

inhibitors2b


3115762.5111533.61311.51111.43733382137.6302825

7.99172697230.95393920147.778553850223.60555127550.23859798830.35694257240.16165807540.50.88573886292.11660104890.71103650870.41508031355.36055967230.86694867214.89672339434.43030472996.55743852432.6457513111

19-Jul


av31111533.61311.51111.437332825

SD7.991726972323.60555127550.23859798830.35694257240.16165807540.50.88573886292.11660104890.71103650870.41508031356.55743852432.6457513111



1570.9539392014

62.57.7785538502

112

153.6055512755

48h

cell extract



317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract



317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract



1570.9539392014

62.57.7785538502

112

153.6055512755

48h

cell extract



317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract


1 EL p50NFkB

1570.9539392014

62.57.7785538502

112

153.6055512755

48h

cell extract


EL p50 controls

317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract



1570.9539392014

62.57.7785538502

112

153.6055512755

48h

cell extract



317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract



1570.9539392014

62.57.7785538502

112

153.6055512755

48h

cell extract


C3 protein wykres

C3 protein wykres

0.280.0115470054

0.370.0251661148

0.40.0057735027

0.530.0057735027

0.0260.0005773503

0.0240.0005773503

0.5450.0025166115

-0.010.0005773503

30min

nuclear extract

OD value [450nm]

inhibitors 2A

0.280.0115470054

0.370.0251661148

0.40.0057735027

0.530.0057735027

0.0260.0005773503

0.0240.0005773503

0.5450.0025166115

-0.010.0005773503

30min

nuclear extract

OD value [450nm]

C3 proteimna graph or

471.5275252317

242.5166114784

221.443375673

4.90.1527525232

6.20.2645751311

4.250.2306512519

38.31.5275252317

7.10.3511884584

6.90.2

48h

cell extract


C3 proteina

C3 proteina

471.5275252317

242.5166114784

221.443375673

4.90.1527525232

6.20.2645751311

4.250.2306512519

38.31.5275252317

7.10.3511884584

6.90.2

48h

cell extract


1NFkB ELISA

471.5275252317

242.5166114784

221.443375673

4.90.1527525232

6.20.2645751311

4.250.2306512519

38.31.5275252317

7.10.3511884584

6.90.2

48h

cell extract





average4724224.256.24.96.97.138.3

14822213.946.64.76.57.236

2491918.54.46.14.96.36.837

34624214.26.54.66.16.539

average47.522.2520.6254.186.44.73333333336.36.833333333337.3333333333

SD1.52752523172.51661147841.4433756730.23065125190.26457513110.15275252320.20.35118845841.5275252317

Br52/2


average4724224.96.24.2538.37.16.9

14822214.76.63.94367.26.5

2491918.54.96.14.4376.86.3

34624214.66.54.2396.56.1

average47.522.2520.6254.73333333336.44.1837.33333333336.83333333336.3

SD1.52752523172.51661147841.4433756730.15275252320.26457513110.23065125191.52752523170.35118845840.2

0.280.0115470054

0.370.0251661148

0.40.0057735027

0.530.0057735027

0.0260.0005773503

0.0240.0005773503

0.5450.0025166115

-0.010.0005773503

30min

nuclear extract

OD value [450nm]


average0.2170.0330.2040.1240.1560.0620.047

10.2170.2170.1240.160.060.047

20.2140.0360.2080.1210.150.0610.044

30.220.0330.2040.1250.1620.0640.049

average0.2170.03450.20966666670.12333333330.15733333330.06166666670.0466666667

SD0.0030.00212132030.00665832810.0020816660.00642910050.0020816660.0025166115


av0.20.30.350.530.0270.0240.545-0.01

0.30.40.40.530.0260.0230.54-0.01

0.280.350.390.520.0250.0240.542-0.011

av0.260.350.380.52666666670.0260.02366666670.5423333333-0.0103333333

SD0.05291502620.050.02645751310.00577350270.0010.00057735030.00251661150.0005773503

Ttest0.00156259920.00035917490.00002060.00000000.00000000.0000007


av0.280.370.40.530.0260.0240.545-0.01

0.30.40.40.530.0260.0230.54-0.01

0.280.350.390.520.0250.0240.542-0.011

av0.28666666670.37333333330.39666666670.52666666670.02566666670.02366666670.5423333333-0.0103333333

SD0.01154700540.02516611480.00577350270.00577350270.00057735030.00057735030.00251661150.0005773503

Ttest0.00000255570.00001811070.00000000.00000000.00000000.0000002



24h1410.672.90.580.4931.170.96.30.630.69

13.70.90.592.40.550.512.61.280.85.80.90.77

15.21.10.712.20.610.522.91.41.15.70.860.82

av14.310.65666666672.50.580.50666666672.83333333331.28333333330.93333333335.93333333330.79666666670.76

SD0.79372539330.10.06110100930.36055512750.030.01527525230.20816659990.11503622620.15275252320.32145502540.145716620.0655743852

test

3d15.540.6560.783.50.40.93.12.11.86.70.780.5

16.40.70.793.40.520.913.22.21.66.40.980.6

16.30.630.822.950.490.893.52.31.56.20.890.67

av16.080.6620.79666666673.28333333330.470.93.26666666672.21.63333333336.43333333330.88333333330.59

SD0.47031904070.0353836120.020816660.29297326390.062449980.010.20816659990.10.15275252320.25166114780.1001665280.0854400375

SD


24h14.310.65666666672.50.580.50666666672.83333333331.28333333330.93333333335.93333333330.79666666670.76

3d16.080.6620.79666666673.28333333330.470.93.26666666672.21.63333333336.43333333330.88333333330.59

SD240.79372539330.10.06110100930.36055512750.030.01527525230.20816659990.11503622620.15275252320.32145502540.145716620.0655743852

SD3d0.47031904070.0353836120.020816660.29297326390.062449980.010.20816659990.10.15275252320.25166114780.1001665280.0854400375

14.316.08350.79372539330.47031904071

10.6620.93333333330.10.0353836120.1527525232

0.65666666670.79666666670.96333333330.06110100930.020816660.118462371

2.53.28333333333.33333333330.36055512750.29297326390.4509249753

0.580.470.75666666670.030.062449980.0585946528

0.50666666670.90.80.01527525230.010.1

2.83333333333.26666666674.10.20816659990.20816659990.1732050808

1.28333333332.22.46666666670.11503622620.10.2516611478

0.93333333331.63333333332.09333333330.15275252320.15275252320.1101514109

5.93333333336.433333333311.40.32145502540.25166114781.1532562595

0.79666666670.88333333330.91333333330.145716620.1001665282.74

0.760.590.13233333330.06557438520.08544003750.0924463809

24h

3d

7d

cell extract



0.79372539330.4703190407

0.10.035383612

0.06110100930.02081666

0.36055512750.2929732639

0.030.06244998

0.01527525230.01

0.20816659990.2081665999

0.11503622620.1

0.15275252320.1527525232

0.32145502540.2516611478

0.145716620.100166528

0.06557438520.0854400375

24h

3d

cell extract


135.33333333336.1101009266

654.3588989435

66.33333333331.5275252317

5.56666666670.5131601439

5.50.5

5.53333333330.9237604307

100.333333333317.616280349

1118.1853527719

85.33333333338.9628864398

33.33333333334.9328828623

37.66666666678.5049005481

33.333333333310.4083299973

214.3588989435

21.66666666678.9628864398

21.66666666678.0829037687

53.66666666675.5075705473

51.66666666676.6583281185

495.2915026221

65.666666666711.0151410946

47.33333333332.5166114784

492.6457513111

3.66666666670.5773502692

5.33333333330.5773502692

3.66666666670.5773502692

48h

cell extract


135.33333333336.1101009266

654.3588989435

66.33333333331.5275252317

5.56666666670.5131601439

5.50.5

5.53333333330.9237604307

100.333333333317.616280349

1118.1853527719

85.33333333338.9628864398

33.33333333334.9328828623

37.66666666678.5049005481

33.333333333310.4083299973

214.3588989435

21.66666666678.9628864398

21.66666666678.0829037687

53.66666666675.5075705473

51.66666666676.6583281185

495.2915026221

65.666666666711.0151410946

47.33333333332.5166114784

492.6457513111

3.66666666670.5773502692

5.33333333330.5773502692

3.66666666670.5773502692

48h

cell extract


141.66512.66

108.66457.7

10.3387.33

19.16191.66

20.76237

0.311.5

0.2410.5

3d

7d

supernatants

C3 Protein [ng/ml]

141.66512.66

108.66457.7

10.3387.33

19.16191.66

20.76237

0.311.5

0.2410.5

3d

7d

supernatants

C3 Protein [ng/ml]


135.33333333336566.33333333335.56666666675.55.5333333333100.333333333311185.333333333333.333333333337.666666666733.33333333332121.666666666721.666666666753.666666666751.66666666674965.666666666747.3333333333493.66666666675.33333333333.6666666667Average

6.11010092664.35889894351.52752523170.51316014390.50.923760430717.6162803498.18535277198.96288643984.93288286238.504900548110.40832999734.35889894358.96288643988.08290376875.50757054736.65832811855.291502622111.01514109462.51661147842.64575131110.57735026920.57735026920.5773502692SD

0.00008431090.00001958870.00001947480.00002075310.02798383280.00100840240.024151360.00113383070.00774020590.00827556490.00024601170.00122031550.04907193280.01557401090.00690512192.51661147840.0055620.00001740970.00001942550.0000174097Ttest

135.33333333336.1101009266

37916.8226038413

654.3588989435

66.33333333331.5275252317

5.56666666670.5131601439

5.50.5

5.53333333330.9237604307

100.333333333317.616280349

1118.1853527719

85.33333333338.9628864398

33.33333333334.9328828623

37.66666666678.5049005481

33.333333333310.4083299973

214.3588989435

21.66666666678.9628864398

21.66666666678.0829037687

53.66666666675.5075705473

51.66666666676.6583281185

495.2915026221

65.666666666711.0151410946

47.33333333332.5166114784

492.6457513111

3.66666666670.5773502692

5.33333333330.5773502692

3.66666666670.5773502692

48h

cell extract


141.66512.66

108.66457.7

10.3387.33

19.16191.66

20.76237

0.311.5

0.2410.5

3d

7d

supernatants

C3 Protein [ng/ml]

PROTEINAVERAGESD

IL-1141.66666677.637626158

TNF-alfa108.66666673.214550254

HIV [0.5]10.333333331.040833

HIV [1pg]19.166666672.020725942

HIV [2.5pg]20.766666671.078579312

mock0.30.2

medium0.2433333330.049328829

IL-1512.66666676.429100507

TNF-alfa457.76666678.494900431

HIV [0.5]87.333333333.214550254

HIV [1pg]191.66333336.345079458

HIV [2.5pg]236.666666712.58305739

mock3.510.5

medium30.1


AV3d141.66108.6610.3319.1620.760.30.24

Av7d512.66457.787.33191.6623711.510.5

SD3d7.633.21.042.021.070.20.04

SD7d6.428.43.26.312.53.52


0.20.30.350.530.0270.0240.545-0.01

0.220.320.360.550.0230.0220.546-0.011

0.210.330.3670.60.0210.0230.546-0.012

av

SD

MBD0009EEC5.xls

22,3

53

24

50

5.65

2.5

1.4

0.5

25

22

22.5

1-control(+)HIV, 2-Control(-)HIV, 3-Calphostin-C, 4-SQ22536, 5-CAPE 1:10000, 6-CAPE 1:15000, 7-Genistein 1:1000, 8-Genistein 1:2000, 9-PMA, 10-Pertusis toxin, 11-8Br-cAMP

inhibitors

RATIO pGLC3pro/pRLTK dual luciferase assay

Br22/3 C3 PROMOTER INDUCTION by HIV with INHIBITORS

22,3+-

5312

2421

5033

5.654.5

2.54.6

1.40.98

0.50.4

2522

2215

22.515

contr(+)/(-)

Calph

SQ22536

CAPE:10000

CAPE;15000

Genist:1000

Genist:2000

PMA

Pertusis toxin

8Br-cAMP

+HIV

-HIV

inhibitors with/w-o HIV

RATIO pGLC3pro/pRLTK Dual Luciferase Assay

BRAIN 22/3

Sheet1

5312

2421

5033

5.654.5

2.54.6

1.40.98

0.50.4

2522

2215

22.515

Sheet1

0

0

0

0

0

0

0

0

0

0

0

inhibitors(-)HIV

inhibitors

Ratio pGlC3/pRLTH

C3 induction promoter - inhibitors without HIV

Br22,4

Br22,4

49

25

16.5

44

18

7.6

4.7

1.2

20

22

31.4

35

Br22/4 inhibitors (+) HIV

inhibitors

Ratio pGL3C/pRLTK

-

20

22.5

30

33.5

11

9.7

1.3

3.4

20

20

32

25

inhibitors(+) HIV

inhibitors

Ratio pGLC3/pRLTK Dual-Luciferase Assay

BR 22/4 C3 induction promoter

49

25

16.5

44

18

7.6

4.7

1.2

20

22

31.4

35

inhibitors (-) HIV

18,4

50

46

26

42

18.4

126

84

72.5

75.6

inhibitors (+) HIV

inhibitors

Ratio pGLC3/pRLTK

Br 18/5 C3 induction promoter

18,4 bez HIV

23

42

35

56

16

72

45

80

38

inhibitors (-) HIV

br4p4

4920

2522.5

16.530

4433.5

1811

7.69.7

4.71.3

1.23.4

2020

2220

31.432

3525

control(+)/(-)

Calph:1000

Calph10-5

SQ22536

CAPE:20000

CAPE:15000

Genist:2000

Genist:1000

8-Br-cAMP(1pgHIV)

PMA(1pgHIV)

Pertusis t1x

Pertusis t5x

(+)HIV

(-)HIV


RATIO pGLC3pro/pRLTK

Brain 22/4

C 3 promoter induction March06

7.5

4.5

2.5

3.8

1

cell extract

Fold change [Relative Luciferase Unit

C3 promoter induction by HIV in HFA

MVP and C3promoter

7.5

4.5

2.5

3.8

1

cell extract

Fold change [Luciferase Activity Unit]

C3 promoter induction by HIV[MVP] in HFA

Sheet14

IL1-beta[10ng/ml]TNF[10U/ml]MVP[2pgP24/cell]MVP 5pgP24/cellcontrol ext.

7.54.52.53.81

18,4por

5023

4642

2635

4256

18.416

12672

8445

72.580

75.638

control(+)/(-)

Calph

CAPE :20000

CAPE :50000

Genist:2000

8-Br-c-AMP(0.5 pg ADA)

PMA

SQ22536

Pertusis toxin

(+)HIV

(-)HIV



BRAIN 18/5

oct11

4421

3515

7737

1812

6045.6

3833

3016.6

1816

4641

1.61

2.51.6

2515

+HIV/HIV-

+HIV

Daid.

Wort.

PDTC

U126

SB202190

SB203580

SP

PT

CAPE

Gen

Calp

inhibitors with/w-oHIV

Ratio pGLC3pro/RLTK

Brain22/4 inhib part1and2 October11

br22part2inh

4421

3515

7737

1812

6045.6

3833

3016.6

1816

4641

1.61

2.51.6

2515

Sheet2

492050237012

2522.546427030

16.5302635112171

4433.542562.42

181118.41645

7.69.7126728488

4.71.3844514090

1.23.472.580113150

202075.638147130

22202643

31.4325158

35254072

82107

107262

159130

45104

6451

Sheet2

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

+HIV

-HIV

inhibitors with/or without HIV

RATIO pGLC3/RLTK

BRAIN 22/3 # NOV12

Il1 beta Jims

160

126

125

Jims stock U/ml

Influence of IL1-beta on induction C3 promoter

C 3 gene expr 48h

8.68

12.3

1

0.6

48h

cell extract

Fold change

C3 gene expression in HFA by HIV

Mouse signal transduction study

217

130

140

38

60

47

227

206

100

11

14

11

17

15

20

59

80

78

340

cell extract

Fold change

Effect of signal transduction's inhibitors on C3 induction promoter by HIV in primary mouse astrocytes

C3 promoter and mouse 2

238

152

138

1.5

1.3

46

35

20

44

17.5

23

25

80

122

104

173

193

227

340

cell extract

Fold change

Effect of signal transduction's inhibitors on C3 promoter induction by HIV in primary mouse astrocytes

Sheet11

NDKcontrol extmediumSB 190/NDKSB190/control extSB190/mediumCalphostion/NDKCalphostin/control ext.Calphostin/mediumJNK inh/NDKJNKinh/cont.extJNK/mediumPDTC/NDKPDTC/control ext.PDTC/mediumSB580/NDKSB580/control extSB580/mediumIL/TNF

217130140386047227206100111411171520598078340

NDKcontrol extmediumCape/NDKCape/control extCape/mediumGenistein/NDKGenistein/control extGenistein/mediumPD 98/NDKPD98/control extPD 98/mediumAG18/NDKAG18/control extAG18/mediump38/NDKp38/control extp38/mediumIL/TNF

2381521381.51.34635204417.5232580122104173193227340

mouse and inhibitors

217.5

130

140

SB190

38

60

47

SB580

59

80

78

Calphostin

227

206

100

JNK

11

14

11

PDTC

17

15

20

SQ

130

170

144

Pertusis toxin

236

225

205

PD 98

17.5

23

25

AG18

80

122

104

P38

173

193

227

MFA

cell extract

Relative Luciferase Activity [RLA]

Effect of signal transduction inhibitors on C3 promoter induction by HIV in primary mouse astrocytes

Sheet10

NDK217.5

c.ext130

medium140

SB190

+ NDK38

c.ext60

medium47

SB580

+ NDK59

c.ext80

medium78

Calphostin

+ NDK227

c.ext206

medium100

JNK

+ NDK11

c.ext14

medium11

PDTC

+ NDK17

c.ext15

medium20

SQ

+ NDK130

c.ext170

medium144

Pertusis toxin

+ NDK236

c.ext225

medium205

PD 98

+ NDK17.5

c.ext23

medium25

AG18

+ NDK80

c.ext122

medium104

P38

+ NDK173

c.ext193

medium227

gene expr 7 days

37.6

3378.5

1

0.84

7 days

cell extract

Fold change

C 3 gene expression in HFA by HIV

Sheet9

MVP[5pgP24/cellIL1-beta[10ng/ml]control extractmedium

37.63378.510.84

MVP[5pgP24/cell]IL1-beta[10ng/ml]control extractmedium

8.6812.310.6

Sheet8

1.0430.1891.0150.545

C3 gene exp 7days

4.84

37.6

1

376.7

3378.6

7 days

cell extract

fold change

C3 gene expression by HIV {MVP} in HFA on 7 days of infection

Sheet7

7days

MVP [J]1pg5pgc.extTNFIL1

4.8437.61376.73378.6

Sheet4

12negative16020U/ml13720ng/ml138

10720012610U/ml11910ng/ml119

601001250.2U/ml1382ng/ml137

5650

Sheet4

0

0

0

0

-/+HIV

Ratio pGLC3pro/RLTK

Induction of C3 promoter by HIV dose dependent

C3 gene expr march24

0

0

0

Jim's stock

Il 1-beta [U/ml]

Ratio pGLC3 pro/RLTK

Influence of IL1-beta on C3 induction promoter

Sheet13

0

0

0

New Il1-beta

IL1-beta [ng/ml]

Ratio pGLC3pro/RLTK

Influence of Il1-beta on C3 induction promoter

p50 NFkB BR37

1.68

2.82

2.44

3.67

1

1.04

24h

Cell extract

Fold change

C3 gene expresion in HFA by HIV

p50-p65p

Panomic dec12

0

00.2

00.3

00.35

00.53

00.027

00.024

00.545

0-0.01

cell extract

OD [p50-p65]

Detection of p50 subunits of heterodimer p50-p65 NFkB in HFA after HIV infection

Sheet12

0.2

0.3

0.35

0.53

0.027

0.024

0.545

-0.01

OD [NFkBp50]

Sheet6

0.20.0529150262

0.30.05

0.350.0264575131

0.530.0057735027

0.0270.001

0.0240.0005773503

0.5450.0025166115

-0.010.0005773503

**

***

****

****

****

cell extract

OD [NFkB subunit p50]

Sheet5


av0.20.30.350.530.0270.0240.545-0.01

0.30.40.40.530.0260.0230.54-0.01

0.280.350.390.520.0250.0240.542-0.011

av0.260.350.380.52666666670.0260.02366666670.5423333333-0.0103333333

SD0.05291502620.050.02645751310.00577350270.0010.00057735030.00251661150.0005773503

Ttest0.00156259920.00035917490.00002060.00000000.00000000.0000007

NDK

Sheet3

NDK fNDK nNL4-3 fIL/TNFc.ext 10ulmedium

1.682.822.443.6711.04

3.63.491

1.60.7614

5081

67120

6040

116.665

67.7

0

0

0

0

0

0

0

Cape exp

inhibitor/+-IL1beta/+-HIV

Ratio pGLC3pro/RLTK

CAPE/+-HIV/+-IL1beta

0

0

0

0

0

0

GENISTEIN exp

inhibitor/+-HIV/+_ IL1-beta

RATIO pGLC3pro/RLTK

GENISTEIN/+-HIV/+_IL1-beta

0

0

Controls- HFA brain28/3 infection NL4-3/PBL

HIV/-HIV

RATIO pGLC3pro/RLTK

149

2430

148.5184.4

119.25210.25

81153

52.2573.33

120206.66

14.6616.33

2741

96152.33

59.33125.33

71.6670

00

2438

00

00

00

00

00

00

00

Ratio

Effect of HIV on the activity of different C3 promoter constructs in HFA

00

00

00

00

00

00

00

Ratio

Effect of HIV on the activity of different C3 promoter constructs in HFA [systemA/2]

MBD0009EEC5.xls

22,3

53

24

50

5.65

2.5

1.4

0.5

25

22

22.5


inhibitors



22,3+-

5312

2421

5033

5.654.5

2.54.6

1.40.98

0.50.4

2522

2215

22.515

contr(+)/(-)

Calph

SQ22536

CAPE:10000

CAPE;15000

Genist:1000

Genist:2000

PMA

Pertusis toxin

8Br-cAMP

+HIV

-HIV



BRAIN 22/3

Sheet1

5312

2421

5033

5.654.5

2.54.6

1.40.98

0.50.4

2522

2215

22.515

Sheet1

0

0

0

0

0

0

0

0

0

0

0

inhibitors(-)HIV

inhibitors

Ratio pGlC3/pRLTH


Br22,4

Br22,4

49

25

16.5

44

18

7.6

4.7

1.2

20

22

31.4

35


inhibitors

Ratio pGL3C/pRLTK

-

20

22.5

30

33.5

11

9.7

1.3

3.4

20

20

32

25

inhibitors(+) HIV

inhibitors



49

25

16.5

44

18

7.6

4.7

1.2

20

22

31.4

35

inhibitors (-) HIV

18,4

50

46

26

42

18.4

126

84

72.5

75.6

inhibitors (+) HIV

inhibitors

Ratio pGLC3/pRLTK


18,4 bez HIV

23

42

35

56

16

72

45

80

38

inhibitors (-) HIV

br4p4

4920

2522.5

16.530

4433.5

1811

7.69.7

4.71.3

1.23.4

2020

2220

31.432

3525

control(+)/(-)

Calph:1000

Calph10-5

SQ22536

CAPE:20000

CAPE:15000

Genist:2000

Genist:1000

8-Br-cAMP(1pgHIV)

PMA(1pgHIV)

Pertusis t1x

Pertusis t5x

(+)HIV

(-)HIV



Brain 22/4


7.5

4.5

2.5

3.8

1

cell extract



MVP and C3promoter

7.5

4.5

2.5

3.8

1

cell extract



Sheet14

IL1-beta[10ng/ml]TNF[10U/ml]MVP[2pgP24/cell]MVP 5pgP24/cellcontrol ext.

7.54.52.53.81

18,4por

5023

4642

2635

4256

18.416

12672

8445

72.580

75.638

control(+)/(-)

Calph

CAPE :20000

CAPE :50000

Genist:2000

8-Br-c-AMP(0.5 pg ADA)

PMA

SQ22536

Pertusis toxin

(+)HIV

(-)HIV



BRAIN 18/5

oct11

4421

3515

7737

1812

6045.6

3833

3016.6

1816

4641

1.61

2.51.6

2515

+HIV/HIV-

+HIV

Daid.

Wort.

PDTC

U126

SB202190

SB203580

SP

PT

CAPE

Gen

Calp

inhibitors with/w-oHIV

Ratio pGLC3pro/RLTK

Brain22/4 inhib part1and2 October11

br22part2inh

4421

3515

7737

1812

6045.6

3833

3016.6

1816

4641

1.61

2.51.6

2515

Sheet2

492050237012

2522.546427030

16.5302635112171

4433.542562.42

181118.41645

7.69.7126728488

4.71.3844514090

1.23.472.580113150

202075.638147130

22202643

31.4325158

35254072

82107

107262

159130

45104

6451

Sheet2

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

+HIV

-HIV

inhibitors with/or without HIV

RATIO pGLC3/RLTK

BRAIN 22/3 # NOV12

Il1 beta Jims

160

126

125

Jims stock U/ml

Influence of IL1-beta on induction C3 promoter

C 3 gene expr 48h

8.68

12.3

1

0.6

48h

cell extract

Fold change

C3 gene expression in HFA by HIV

Mouse signal transduction study

217

130

140

38

60

47

227

206

100

11

14

11

17

15

20

59

80

78

340

cell extract

Fold change

Effect of signal transduction's inhibitors on C3 induction promoter by HIV in primary mouse astrocytes

C3 promoter and mouse 2

238

152

138

1.5

1.3

46

35

20

44

17.5

23

25

80

122

104

173

193

227

340

cell extract

Fold change

Effect of signal transduction's inhibitors on C3 promoter induction by HIV in primary mouse astrocytes

Sheet11

NDKcontrol extmediumSB 190/NDKSB190/control extSB190/mediumCalphostion/NDKCalphostin/control ext.Calphostin/mediumJNK inh/NDKJNKinh/cont.extJNK/mediumPDTC/NDKPDTC/control ext.PDTC/mediumSB580/NDKSB580/control extSB580/mediumIL/TNF

217130140386047227206100111411171520598078340

NDKcontrol extmediumCape/NDKCape/control extCape/mediumGenistein/NDKGenistein/control extGenistein/mediumPD 98/NDKPD98/control extPD 98/mediumAG18/NDKAG18/control extAG18/mediump38/NDKp38/control extp38/mediumIL/TNF

2381521381.51.34635204417.5232580122104173193227340

mouse and inhibitors

217.5

130

140

SB190

38

60

47

SB580

59

80

78

Calphostin

227

206

100

JNK

11

14

11

PDTC

17

15

20

SQ

130

170

144

Pertusis toxin

236

225

205

PD 98

17.5

23

25

AG18

80

122

104

P38

173

193

227

MFA

cell extract

Relative Luciferase Activity [RLA]

Effect of signal transduction inhibitors on C3 promoter induction by HIV in primary mouse astrocytes

Sheet10

NDK217.5

c.ext130

medium140

SB190

+ NDK38

c.ext60

medium47

SB580

+ NDK59

c.ext80

medium78

Calphostin

+ NDK227

c.ext206

medium100

JNK

+ NDK11

c.ext14

medium11

PDTC

+ NDK17

c.ext15

medium20

SQ

+ NDK130

c.ext170

medium144

Pertusis toxin

+ NDK236

c.ext225

medium205

PD 98

+ NDK17.5

c.ext23

medium25

AG18

+ NDK80

c.ext122

medium104

P38

+ NDK173

c.ext193

medium227

gene expr 7 days

37.6

3378.5

1

0.84

7 days

cell extract

Fold change

C 3 gene expression in HFA by HIV

Sheet9

MVP[5pgP24/cellIL1-beta[10ng/ml]control extractmedium

37.63378.510.84

MVP[5pgP24/cell]IL1-beta[10ng/ml]control extractmedium

8.6812.310.6

Sheet8

1.0430.1891.0150.545

C3 gene exp 7days

4.84

37.6

1

376.7

3378.6

7 days

cell extract

fold change

C3 gene expression by HIV {MVP} in HFA on 7 days of infection

Sheet7

7days

MVP [J]1pg5pgc.extTNFIL1

4.8437.61376.73378.6

Sheet4

12negative16020U/ml13720ng/ml138

10720012610U/ml11910ng/ml119

601001250.2U/ml1382ng/ml137

5650

Sheet4

0

0

0

0

-/+HIV

Ratio pGLC3pro/RLTK

Induction of C3 promoter by HIV dose dependent

C3 gene expr march24

0

0

0

Jim's stock

Il 1-beta [U/ml]

Ratio pGLC3 pro/RLTK

Influence of IL1-beta on C3 induction promoter

Sheet13

0

0

0

New Il1-beta

IL1-beta [ng/ml]

Ratio pGLC3pro/RLTK

Influence of Il1-beta on C3 induction promoter

p50 NFkB BR37

1.68

2.82

2.44

3.67

1

1.04

24h

Cell extract

Fold change

C3 gene expresion in HFA by HIV

p50-p65p

Panomic dec12

0

00.2

00.3

00.35

00.53

00.027

00.024

00.545

0-0.01

cell extract

OD [p50-p65]

Detection of p50 subunits of heterodimer p50-p65 NFkB in HFA after HIV infection

Sheet12

0.2

0.3

0.35

0.53

0.027

0.024

0.545

-0.01

OD [NFkBp50]

Sheet6

0.20.0529150262

0.30.05

0.350.0264575131

0.530.0057735027

0.0270.001

0.0240.0005773503

0.5450.0025166115

-0.010.0005773503

**

***

****

****

****

cell extract

OD [NFkB subunit p50]

Sheet5


av0.20.30.350.530.0270.0240.545-0.01

0.30.40.40.530.0260.0230.54-0.01

0.280.350.390.520.0250.0240.542-0.011

av0.260.350.380.52666666670.0260.02366666670.5423333333-0.0103333333

SD0.05291502620.050.02645751310.00577350270.0010.00057735030.00251661150.0005773503

Ttest0.00156259920.00035917490.00002060.00000000.00000000.0000007

NDK

Sheet3

NDK fNDK nNL4-3 fIL/TNFc.ext 10ulmedium

1.682.822.443.6711.04

3.63.491

1.60.7614

5081

67120

6040

116.665

67.7

0

0

0

0

0

0

0

Cape exp

inhibitor/+-IL1beta/+-HIV

Ratio pGLC3pro/RLTK

CAPE/+-HIV/+-IL1beta

0

0

0

0

0

0

GENISTEIN exp

inhibitor/+-HIV/+_ IL1-beta

RATIO pGLC3pro/RLTK

GENISTEIN/+-HIV/+_IL1-beta

0

0

Controls- HFA brain28/3 infection NL4-3/PBL

HIV/-HIV

RATIO pGLC3pro/RLTK

149

2430

148.5184.4

119.25210.25

81153

52.2573.33

120206.66

14.6616.33

2741

96152.33

59.33125.33

71.6670

00

2438

00

00

00

00

00

00

00

Ratio

Effect of HIV on the activity of different C3 promoter constructs in HFA

00

00

00

00

00

00

00

Ratio

Effect of HIV on the activity of different C3 promoter constructs in HFA [systemA/2]

MBD0003B060.doc

MBD000020EA.xls

C3protein wykres or

141.66512.667.66.4

108.66457.73.28.4

10.3387.331.043.2

19.16191.6626.3

20.762371.0712.5

0.311.533

0.2410.5

C

3d

7d

supernatants

C3 Protein [ng/ml]

inhibitors 2b graph

317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract


graph cont inhib 2b

1577.9917269723

62.52

113.6055512755

150.2385979883

48h

cell extract




average31111515762.51115

25.51316156.5551316

40.011118158.170.531118

26.95911156.461.97911

average30.82111515762.51115

SD7.991726972323.60555127550.95393920147.778553850223.6055512755

Ttest0.03534436910.00000031680.0006591335


15762.51115

156.5551316

158.170.531118

156.461.97911

av15762.51115

SD0.95393920147.778553850223.6055512755

0.0006591335

inhibitors2b


3115762.5111533.61311.51111.43733382137.6302825

7.99172697230.95393920147.778553850223.60555127550.23859798830.35694257240.16165807540.50.88573886292.11660104890.71103650870.41508031355.36055967230.86694867214.89672339434.43030472996.55743852432.6457513111

19-Jul


av31111533.61311.51111.437332825

SD7.991726972323.60555127550.23859798830.35694257240.16165807540.50.88573886292.11660104890.71103650870.41508031356.55743852432.6457513111



1570.9539392014

62.57.7785538502

112

153.6055512755

48h

cell extract



317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract



317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract



1570.9539392014

62.57.7785538502

112

153.6055512755

48h

cell extract



317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract


1 EL p50NFkB

1570.9539392014

62.57.7785538502

112

153.6055512755

48h

cell extract


EL p50 controls

317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract



1570.9539392014

62.57.7785538502

112

153.6055512755

48h

cell extract



317.9917269723

112

153.6055512755

30.2385979883

3.60.3569425724

130.1616580754

11.50.5

110.8857388629

11.42.1166010489

370.7110365087

330.4150803135

286.5574385243

252.6457513111

48h

cell extract



1570.9539392014

62.57.7785538502

112

153.6055512755

48h

cell extract


C3 protein wykres

C3 protein wykres

0.280.0115470054

0.370.0251661148

0.40.0057735027

0.530.0057735027

0.0260.0005773503

0.0240.0005773503

0.5450.0025166115

-0.010.0005773503

30min

nuclear extract

OD value [450nm]

inhibitors 2A

0.280.0115470054

0.370.0251661148

0.40.0057735027

0.530.0057735027

0.0260.0005773503

0.0240.0005773503

0.5450.0025166115

-0.010.0005773503

30min

nuclear extract

OD value [450nm]

C3 proteimna graph or

471.5275252317

242.5166114784

221.443375673

4.90.1527525232

6.20.2645751311

4.250.2306512519

38.31.5275252317

7.10.3511884584

6.90.2

48h

cell extract


C3 proteina

C3 proteina

471.5275252317

242.5166114784

221.443375673

4.90.1527525232

6.20.2645751311

4.250.2306512519

38.31.5275252317

7.10.3511884584

6.90.2

48h

cell extract


1NFkB ELISA

471.5275252317

242.5166114784

221.443375673

4.90.1527525232

6.20.2645751311

4.250.2306512519

38.31.5275252317

7.10.3511884584

6.90.2

48h

cell extract





average4724224.256.24.96.97.138.3

14822213.946.64.76.57.236

2491918.54.46.14.96.36.837

34624214.26.54.66.16.539

average47.522.2520.6254.186.44.73333333336.36.833333333337.3333333333

SD1.52752523172.51661147841.4433756730.23065125190.26457513110.15275252320.20.35118845841.5275252317

Br52/2


average4724224.96.24.2538.37.16.9

14822214.76.63.94367.26.5

2491918.54.96.14.4376.86.3

34624214.66.54.2396.56.1

average47.522.2520.6254.73333333336.44.1837.33333333336.83333333336.3

SD1.52752523172.51661147841.4433756730.15275252320.26457513110.23065125191.52752523170.35118845840.2

0.280.0115470054

0.370.0251661148

0.40.0057735027

0.530.0057735027

0.0260.0005773503

0.0240.0005773503

0.5450.0025166115

-0.010.0005773503

30min

nuclear extract

OD value [450nm]


average0.2170.0330.2040.1240.1560.0620.047

10.2170.2170.1240.160.060.047

20.2140.0360.2080.1210.150.0610.044

30.220.0330.2040.1250.1620.0640.049

average0.2170.03450.20966666670.12333333330.15733333330.06166666670.0466666667

SD0.0030.00212132030.00665832810.0020816660.00642910050.0020816660.0025166115


av0.20.30.350.530.0270.0240.545-0.01

0.30.40.40.530.0260.0230.54-0.01

0.280.350.390.520.0250.0240.542-0.011

av0.260.350.380.52666666670.0260.02366666670.5423333333-0.0103333333

SD0.05291502620.050.02645751310.00577350270.0010.00057735030.00251661150.0005773503

Ttest0.00156259920.00035917490.00002060.00000000.00000000.0000007


av0.280.370.40.530.0260.0240.545-0.01

0.30.40.40.530.0260.0230.54-0.01

0.280.350.390.520.0250.0240.542-0.011

av0.28666666670.37333333330.39666666670.52666666670.02566666670.02366666670.5423333333-0.0103333333

SD0.01154700540.02516611480.00577350270.00577350270.00057735030.00057735030.00251661150.0005773503

Ttest0.00000255570.00001811070.00000000.00000000.00000000.0000002



24h1410.672.90.580.4931.170.96.30.630.69

13.70.90.592.40.550.512.61.280.85.80.90.77

15.21.10.712.20.610.522.91.41.15.70.860.82

av14.310.65666666672.50.580.50666666672.83333333331.28333333330.93333333335.93333333330.79666666670.76

SD0.79372539330.10.06110100930.36055512750.030.01527525230.20816659990.11503622620.15275252320.32145502540.145716620.0655743852

test

3d15.540.6560.783.50.40.93.12.11.86.70.780.5

16.40.70.793.40.520.913.22.21.66.40.980.6

16.30.630.822.950.490.893.52.31.56.20.890.67

av16.080.6620.79666666673.28333333330.470.93.26666666672.21.63333333336.43333333330.88333333330.59

SD0.47031904070.0353836120.020816660.29297326390.062449980.010.20816659990.10.15275252320.25166114780.1001665280.0854400375

SD


24h14.310.65666666672.50.580.50666666672.83333333331.28333333330.93333333335.93333333330.79666666670.76

3d16.080.6620.79666666673.28333333330.470.93.26666666672.21.63333333336.43333333330.88333333330.59

SD240.79372539330.10.06110100930.36055512750.030.01527525230.20816659990.11503622620.15275252320.32145502540.145716620.0655743852

SD3d0.47031904070.0353836120.020816660.29297326390.062449980.010.20816659990.10.15275252320.25166114780.1001665280.0854400375

14.316.08350.79372539330.47031904071

10.6620.93333333330.10.0353836120.1527525232

0.65666666670.79666666670.96333333330.06110100930.020816660.118462371

2.53.28333333333.33333333330.36055512750.29297326390.4509249753

0.580.470.75666666670.030.062449980.0585946528

0.50666666670.90.80.01527525230.010.1

2.83333333333.26666666674.10.20816659990.20816659990.1732050808

1.28333333332.22.46666666670.11503622620.10.2516611478

0.93333333331.63333333332.09333333330.15275252320.15275252320.1101514109

5.93333333336.433333333311.40.32145502540.25166114781.1532562595

0.79666666670.88333333330.91333333330.145716620.1001665282.74

0.760.590.13233333330.06557438520.08544003750.0924463809

24h

3d

7d

cell extract



0.79372539330.4703190407

0.10.035383612

0.06110100930.02081666

0.36055512750.2929732639

0.030.06244998

0.01527525230.01

0.20816659990.2081665999

0.11503622620.1

0.15275252320.1527525232

0.32145502540.2516611478

0.145716620.100166528

0.06557438520.0854400375

24h

3d

cell extract


135.33333333336.1101009266

654.3588989435

66.33333333331.5275252317

5.56666666670.5131601439

5.50.5

5.53333333330.9237604307

100.333333333317.616280349

1118.1853527719

85.33333333338.9628864398

33.33333333334.9328828623

37.66666666678.5049005481

33.333333333310.4083299973

214.3588989435

21.66666666678.9628864398

21.66666666678.0829037687

53.66666666675.5075705473

51.66666666676.6583281185

495.2915026221

65.666666666711.0151410946

47.33333333332.5166114784

492.6457513111

3.66666666670.5773502692

5.33333333330.5773502692

3.66666666670.5773502692

48h

cell extract


135.33333333336.1101009266

654.3588989435

66.33333333331.5275252317

5.56666666670.5131601439

5.50.5

5.53333333330.9237604307

100.333333333317.616280349

1118.1853527719

85.33333333338.9628864398

33.33333333334.9328828623

37.66666666678.5049005481

33.333333333310.4083299973

214.3588989435

21.66666666678.9628864398

21.66666666678.0829037687

53.66666666675.5075705473

51.66666666676.6583281185

495.2915026221

65.666666666711.0151410946

47.33333333332.5166114784

492.6457513111

3.66666666670.5773502692

5.33333333330.5773502692

3.66666666670.5773502692

48h

cell extract


141.66512.66

108.66457.7

10.3387.33

19.16191.66

20.76237

0.311.5

0.2410.5

3d

7d

supernatants

C3 Protein [ng/ml]

141.66512.667.66.4

108.66457.73.28.4

10.3387.331.043.2

19.16191.6626.3

20.762371.0712.5

0.311.533

0.2410.5

C

3d

7d

supernatants

C3 Protein [ng/ml]


135.33333333336566.33333333335.56666666675.55.5333333333100.333333333311185.333333333333.333333333337.666666666733.33333333332121.666666666721.666666666753.666666666751.66666666674965.666666666747.3333333333493.66666666675.33333333333.6666666667Average

6.11010092664.35889894351.52752523170.51316014390.50.923760430717.6162803498.18535277198.96288643984.93288286238.504900548110.40832999734.35889894358.96288643988.08290376875.50757054736.65832811855.291502622111.01514109462.51661147842.64575131110.57735026920.57735026920.5773502692SD

0.00008431090.00001958870.00001947480.00002075310.02798383280.00100840240.024151360.00113383070.00774020590.00827556490.00024601170.00122031550.04907193280.01557401090.00690512192.51661147840.0055620.00001740970.00001942550.0000174097Ttest

135.33333333336.1101009266

37916.8226038413

654.3588989435

66.33333333331.5275252317

5.56666666670.5131601439

5.50.5

5.53333333330.9237604307

100.333333333317.616280349

1118.1853527719

85.33333333338.9628864398

33.33333333334.9328828623

37.66666666678.5049005481

33.333333333310.4083299973

214.3588989435

21.66666666678.9628864398

21.66666666678.0829037687

53.66666666675.5075705473

51.66666666676.6583281185

495.2915026221

65.666666666711.0151410946

47.33333333332.5166114784

492.6457513111

3.66666666670.5773502692

5.33333333330.5773502692

3.66666666670.5773502692

48h

cell extract


141.66512.66

108.66457.7

10.3387.33

19.16191.66

20.76237

0.311.5

0.2410.5

3d

7d

supernatants

C3 Protein [ng/ml]

PROTEINAVERAGESD

IL-1141.66666677.637626158

TNF-alfa108.66666673.214550254

HIV [0.5]10.333333331.040833

HIV [1pg]19.166666672.020725942

HIV [2.5pg]20.766666671.078579312

mock0.30.2

medium0.2433333330.049328829

IL-1512.66666676.429100507

TNF-alfa457.76666678.494900431

HIV [0.5]87.333333333.214550254

HIV [1pg]191.66333336.345079458

HIV [2.5pg]236.666666712.58305739

mock3.510.5

medium30.1


AV3d141.66108.6610.3319.1620.760.30.24

Av7d512.66457.787.33191.6623711.510.5

SD3d7.633.21.042.021.070.20.04

SD7d6.428.43.26.312.53.52


0.20.30.350.530.0270.0240.545-0.01

0.220.320.360.550.0230.0220.546-0.011

0.210.330.3670.60.0210.0230.546-0.012

av

SD

MBD0009EEC5.xls

22,3

53

24

50

5.65

2.5

1.4

0.5

25

22

22.5


inhibitors



22,3+-

5312

2421

5033

5.654.5

2.54.6

1.40.98

0.50.4

2522

2215

22.515

contr(+)/(-)

Calph

SQ22536

CAPE:10000

CAPE;15000

Genist:1000

Genist:2000

PMA

Pertusis toxin

8Br-cAMP

+HIV

-HIV



BRAIN 22/3

Sheet1

5312

2421

5033

5.654.5

2.54.6

1.40.98

0.50.4

2522

2215

22.515

Sheet1

0

0

0

0

0

0

0

0

0

0

0

inhibitors(-)HIV

inhibitors

Ratio pGlC3/pRLTH


Br22,4

Br22,4

49

25

16.5

44

18

7.6

4.7

1.2

20

22

31.4

35


inhibitors

Ratio pGL3C/pRLTK

-

20

22.5

30

33.5

11

9.7

1.3

3.4

20

20

32

25

inhibitors(+) HIV

inhibitors



49

25

16.5

44

18

7.6

4.7

1.2

20

22

31.4

35

inhibitors (-) HIV

18,4

50

46

26

42

18.4

126

84

72.5

75.6

inhibitors (+) HIV

inhibitors

Ratio pGLC3/pRLTK


18,4 bez HIV

23

42

35

56

16

72

45

80

38

inhibitors (-) HIV

br4p4

4920

2522.5

16.530

4433.5

1811

7.69.7

4.71.3

1.23.4

2020

2220

31.432

3525

control(+)/(-)

Calph:1000

Calph10-5

SQ22536

CAPE:20000

CAPE:15000

Genist:2000

Genist:1000

8-Br-cAMP(1pgHIV)

PMA(1pgHIV)

Pertusis t1x

Pertusis t5x

(+)HIV

(-)HIV



Brain 22/4


7.5

4.5

2.5

3.8

1

cell extract



MVP and C3promoter

7.5

4.5

2.5

3.8

1

cell extract



Sheet14

IL1-beta

november9san diego poster correctio october 26

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