Cell Systems, Volume 5

Supplemental Information

Systematic Investigation of Multi-TLR

Sensing Identifies Regulators of Sustained

Gene Activation in Macrophages

Bin Lin, Bhaskar Dutta, and Iain D.C. Fraser

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ax m

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Time post TLR stimulation (hr)

Il6 Il1a Nfkbiz Tnf

Figure S1. Related to Figure 1.Differential expression characteristics among TLR induced genes in mouse macrophages.BMDM were treated with 5 nM Lipid A for the time indicated and mRNA expression for Il6, Il1a, Nfkbizand Tnf was measured by quantitative PCR. Data are presented as mean±SD from two independentexperiments.

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IL-6

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R848 PIC P3C LPS

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p40

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R848 PIC P3C LPS

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P3C)(ng/ml)

PIC)(ug/ml)

PIC)(ug/ml)

IL412)p4

0)(ng/ml)

R848)(ng/ml) P3C)(ng/ml) R848)(ng/ml)

IL412)p40 LPS)(ng/ml) LPS)(ng/ml) LPS)(ng/ml)

PIC)(ug/ml)

P3C)(ng/ml)

R848)(n

g/ml)

0 1 10 20 50 0 1 10 20 50 0 1 10 20 50

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IL)6 LPS-(ng/ml) LPS-(ng/ml) LPS-(ng/ml)

IL)6-(n

g/ml)

R848-(ng/ml) P3C-(ng/ml) R848-(ng/ml)

PIC-(ug/ml)

P3C-(ng/ml)

R848-(n

g/ml)

P3C-(ng/ml)

PIC-(ug/ml)

PIC-(ug/ml)

Figure S2. Related to Figure 1.Effects of TLR ligand dose and pairwise ligand combinations on cytokine secretion in macrophages. (a+b) BMDMwere treated with single and pairwise combinations of the indicated TLR ligands, and after 24 hr IL-6 (a) and IL-12p40 (b)were measured by ELISA. Cytokine levels are presented as heatmaps from white (low) to red (high).

Figure S2 continued on next page

Figure S2 continued

(c+d) Single TLR ligand dose responses for BMDM stimulated with R848, poly(I:C), Pam3CSK4, and LPS.After 24 hr of ligand treatment, IL-6 (c) and IL-12p40 (d) secretion levels were measured by ELISA. The fourincreasing doses of each TLR ligand used were the same as shown in panels a and b. The doses selected forsubsequent experiments are highlighted blue in panels a and b. Poly I:C is not a strong inducer of either IL-6 orIL-12p40, and didn’t reach saturation even at double the most commonly used dose. Data are presented asmean±SD of three independent experiments.

Obs

erve

d m

RN

A ex

pres

sion

(fol

d)

Predicted mRNA expression (fold)

0 40 80 120 0 40 80 120 0 40 80 120-10

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Liga

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-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140Expected

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Liga

ndEff

ect1+2

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140Expected

0.63 0.640.86 040

8012

0

PIC

TLR3

TRIF

+

dLPS

TLR4

MyD88/TRIF

1 hr 4 hr 8 hr

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-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140Expected

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ect1+2

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140Expected

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Liga

ndEff

ect1+2

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140Expected

0.99 0.890.96

0.98 0.880.94 040

8012

00

4080

120

+

+

PIC

TLR3

TRIF

PIC

TLR3

TRIF

aP3C

TLR2/1

MyD88

R848

TLR7

MyD88

-10

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ndEff

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ndEff

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-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140Expected

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Liga

ndEff

ect1+2

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140Expected

0.54 0.580.60 040

8012

0

+

bP3C

TLR2/1

MyD88

R848

TLR7

MyD88

-10

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Liga

ndEff

ect1+2

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140Expected

0.78 0.750.64

0.82 0.760.65 040

8012

00

4080

120

+

+

c

P3C

TLR2/1

MyD88

LPS

TLR4

MyD88/TRIF

LPS

TLR4

MyD88/TRIF

R848

TLR7

MyD88

Figure S3 (continued from previous page). Related to Figure 2.

Characteristics of transcriptome-wide non-additivity in mRNA expression for 6 pairwise TLR ligandcombinations. Predicted mRNA expression was calculated by summing the net fold induction of the twosingle ligand stimulations for 9,134 transcripts induced in at least three of the 30 tested single or dual TLRligand conditions (see Methods; Data file S1). Each dot represents one upregulated gene, and the predicted vsobserved mRNA expression values are plotted. Red lines are the diagonal reference for additive expression(where predicted and observed mRNA levels are equivalent). Green lines are fitted to the observed datapoints, with the slope of the fitted line indicated on the bottom right of each panel. a) MyD88 specific ligandcombination R848+P3C. b) MyD88+TRIF ligand combinations P3C+PIC and R848+PIC. c)MyD88+MyD88/TRIF ligand combinations R848+LPS and P3C+LPS. d) TRIF+MyD88/TRIF ligandcombination PIC+LPS. Data were derived from duplicate microarrays (see Methods; Data file S1).

Cluster 2:IR/P but not LR/P

synergy(Socs3, Il1b, Il18,

Oasl1)

Cluster 6:Antagonised,

Myd88 dominant (Cxcl1, Slc13a3,

Saa3)

Cluster 4:LR/P specific

synergy (Il12a, Ccnd2)

Cluster 1:Synergized by IR/P and LR/P(Il1a, Il27, Il6,

Il10, Edn1, Nos2, Lcn2,

Cd40)

Cluster 3:IR or IP specific

synergy(Ccl3, Ccl4,

Cd83, Zc3h12a)

Cluster 5:Antagonised, Trif dominant (Il15, Cxcl10,

Ifit2, Ifit3)

Figure S4. Related to Figure 2.Expression characteristics and dual ligand induced synergy of 282 TLR induced genes. Middle panel:Clustering of 282 transcripts with a DIF ≥ 2 or DIF ≤ -2 under 6 pairwise PAMP combinations at 3 time points.Antagonized/synergized fold DIF values (converted to log2 scale), are indicated by the color gradient ofblue/red respectively, while additive or saturated outcomes are white.

Figure S4 continued on next page

global

-5 .09 0 6.07

RI_1h

RI_4h

RI_8h

IP_1h

IP_4h

IP_8h

RL_1h

RL_4h

RL_8h

PL_1h

PL_4h

PL_8h

IL_1h

IL_4h

IL_8h

RP_1h

RP_4h

RP_8h

PROBE_IDOrder.log2sa.clustering.then.manualSymbolILMN_1227018 1 Il1aILMN_3160750 2 AA467197ILMN_2630045 3 Il27ILMN_3072427 4 Il1rnILMN_2965903 5 HdcILMN_1245710 6 Ccl2ILMN_1238505 7 Il10ILMN_1249336 8 Mmp13ILMN_2959291 9 Upp1ILMN_1244866 10 Gbp5ILMN_1223499 11 Nos2ILMN_2737713 12 Edn1ILMN_2835117 13 Ccl7ILMN_1242406 14 Dusp2ILMN_1252202 15 Tnfaip3ILMN_2502136 16 Cd40ILMN_1243601 17 Il6ILMN_2790382 18 Gfi1ILMN_2959292 19 Upp1ILMN_2959293 20 Upp1ILMN_2642349 21 Slc7a2ILMN_1253874 22 Serpinb2ILMN_2712075 23 Lcn2ILMN_2668510 24 HpILMN_2668509 25 HpILMN_2944824 26 HpILMN_2616028 27 PtgesILMN_2679276 28 Il19ILMN_2721571 29 Slamf1ILMN_1219915 30 Dgat2ILMN_2966386 31 Rcl1ILMN_1254929 32 Rcl1ILMN_1244424 33 Adora2bILMN_2654754 34 PdpnILMN_2710905 35 S100a8ILMN_1222009 36 Gab1ILMN_2763679 37 Sipa1l1ILMN_3096144 38 Stat3ILMN_2854004 39 SpicILMN_3054914 40 Usp18ILMN_2771766 41 Ccl12ILMN_2595732 42 Nfil3ILMN_3161805 43 Ccl12ILMN_2846776 44 Cdkn1aILMN_2449157 45 Tnfsf9ILMN_1215209 46 Clec4eILMN_2714031 47 Errfi1ILMN_1215862 48 Cxcl9ILMN_2718330 49 CishILMN_2636403 50 Csrnp1ILMN_2811154 51 Cxcl2ILMN_2733778 52 Il4i1ILMN_2752624 53 Ccrl2ILMN_1219869 54 Il15ILMN_2702303 55 Ch25hILMN_1244513 56 Gbp3ILMN_2778655 57 Vcam1ILMN_1231600 58 Ptgs2ILMN_3112011 59 Dusp16ILMN_2754148 60 Dusp16ILMN_2630605 61 Fscn1ILMN_3009243 62 Dusp2ILMN_2638491 63 Slamf7ILMN_2628822 64 Oas1gILMN_2618176 65 Socs3ILMN_2737685 66 Mmp13ILMN_2722380 67 Oasl1ILMN_2777498 68 Il1bILMN_2843286 69 Slc28a2ILMN_1233733 70 Hsp90ab1ILMN_2727920 71 TspoILMN_2727921 72 TspoILMN_1215632 73 Marcksl1ILMN_2617162 74 Marcksl1ILMN_2622671 75 Acsl1ILMN_3083163 76 CpILMN_1235735 77 Slco3a1ILMN_1245200 78 Ifi205ILMN_2707870 79 Mx1ILMN_2731989 80 Ext1ILMN_2918977 81 March1ILMN_2716602 82 Lgals9ILMN_2662926 83 Egr1ILMN_2786442 84 PtgesILMN_2646322 85 Samsn1ILMN_2676066 86 Stard7ILMN_2652608 87 SpicILMN_2622983 88 Dusp1ILMN_1228657 89 Fcgr2bILMN_2422743 90 Tor3aILMN_3102376 91 Fcgr2bILMN_2709820 92 Dtx2ILMN_2701891 93 Marcksl1ILMN_2486186 94 Tnfrsf1bILMN_1256257 95 Isg15ILMN_1236517 96 Il18ILMN_1239770 97 Peli1ILMN_2674979 98 FusILMN_2899863 99 TnfILMN_2503129 100 Cd40ILMN_1230586 101 Traf1ILMN_1230157 102 Rnd3ILMN_2662990 103 Xkr8ILMN_1237695 104 PfkpILMN_2900653 105 Gadd45bILMN_1219074 106 Slfn2ILMN_2590638 107 Bcl2l11ILMN_2480682 108 Sgms1ILMN_2907322 109 Dtx2ILMN_3161253 110 Shisa3ILMN_2717667 111 Pim3ILMN_1246650 112 Hsp90ab1ILMN_2659108 113 Ppp4r1ILMN_1213167 114 Eif4g2ILMN_1230152 115 Adamts4ILMN_2722938 116 Ppp1r15aILMN_2865016 117 Cd83ILMN_1253919 118 Ccl3ILMN_3107690 119 Fam177aILMN_2588051 120 Tubb2aILMN_3115796 121 Cd40ILMN_1230143 122 Batf2ILMN_2742861 123 Serpina3fILMN_2873793 124 Mapkbp1ILMN_2634970 125 Ripk2ILMN_2918002 126 Gbp3ILMN_3115162 127 Jak2ILMN_1216972 128 Clec4eILMN_2680076 129 Ifi205ILMN_2857947 130 Trim36ILMN_2678580 131 Cers6ILMN_2722732 132 SrgnILMN_1230853 133 CfbILMN_2856095 134 Zc3h12aILMN_1216818 135 Malt1ILMN_1255648 136 Csf3rILMN_2725188 137 Atp10aILMN_2606072 138 Agfg1ILMN_2976191 139 Stat5aILMN_2789125 140 Stard7ILMN_3034034 141 Fam177aILMN_2852034 142 Hif1aILMN_1249498 143 PlekILMN_2761900 144 BatfILMN_1221048 145 Psat1ILMN_2947568 146 Gadd45aILMN_2649067 147 Irf1ILMN_3155492 148 TapbpILMN_2674475 149 Tarm1ILMN_2704637 150 MdficILMN_2717127 151 Ddx58ILMN_2752873 152 Lgals9ILMN_2649068 153 Irf1ILMN_2705119 154 Btg1ILMN_1238752 155 NfkbieILMN_2905866 156 Cited2ILMN_1224472 157 Ccl4ILMN_2700166 158 Ccnd2ILMN_2589790 159 Stat3ILMN_3103896 160 Timp1ILMN_2769918 161 Timp1ILMN_2751527 162 SrgnILMN_2926198 163 HilpdaILMN_2662802 164 Ptx3ILMN_2676127 165 Scarf2ILMN_1248181 166 Zbtb7aILMN_2698046 167 Stat3ILMN_2648548 168 Casp7ILMN_2600426 169 Ccdc94ILMN_2868220 170 InhbaILMN_2688912 171 F10ILMN_2706462 172 Il12aILMN_2596596 173 Rgs1ILMN_2596970 174 Tmem39aILMN_1246194 175 Ifit3bILMN_1230458 176 Ifi t3ILMN_2981167 177 Ifi t2ILMN_2846865 178 ActbILMN_2588055 179 ActbILMN_1233172 180 Pou3f1ILMN_2981169 181 Ifi t2ILMN_2696491 182 Phf11aILMN_2903945 183 Gadd45gILMN_2793464 184 Oasl1ILMN_1240864 185 Cmpk2ILMN_2684848 186 Phf11dILMN_2696492 187 Phf11aILMN_2717366 188 Oasl1ILMN_2827780 189 Phf11dILMN_2711910 190 Ifnb1ILMN_3142803 191 Cxcl10ILMN_1227573 192 Irf7ILMN_2923615 193 Ppm1kILMN_2944666 194 Ifi t3ILMN_3122961 195 Gbp2ILMN_1230357 196 Itpr1ILMN_1237736 197 Plekhf2ILMN_2845658 198 BC147527ILMN_2743244 199 Cd86ILMN_2744890 200 Gadd45gILMN_1234539 201 Irgm1ILMN_2492716 202 UacaILMN_1259564 203 Irgm2ILMN_1244891 204 Cst7ILMN_1217041 205 Cd86ILMN_1225204 206 Rsad2ILMN_2598622 207 Slamf8ILMN_2652857 208 Ifi47ILMN_2762380 209 Enpp4ILMN_2959253 210 Dnaaf3ILMN_2626939 211 DaxxILMN_2870302 212 Parp3ILMN_2860645 213 Gbp7ILMN_1213978 214 PtproILMN_2829594 215 Hspa1aILMN_1249864 216 Fam26fILMN_2603081 217 Nt5c3ILMN_1239219 218 Mx2ILMN_2725927 219 Serpina3gILMN_2599782 220 Irf1ILMN_2654554 221 Fgl2ILMN_1247710 222 Nod1ILMN_2971721 223 Fbrsl1ILMN_2610234 224 Il15ILMN_2834777 225 Irf1ILMN_1218240 226 Cd69ILMN_2990616 227 Birc2ILMN_2938704 228 Il15ILMN_2902979 229 FasILMN_1250438 230 Marcksl1ILMN_2897891 231 Rgs1ILMN_2618918 232 Slc2a6ILMN_2804166 233 Igsf9ILMN_2690551 234 NfkbieILMN_3148543 235 SrcILMN_2625377 236 Rgs1ILMN_2663374 237 Nr1h3ILMN_1221750 238 MyclILMN_1246609 239 Rasgrp1ILMN_2728729 240 Sdc4ILMN_2628757 241 Ehd1ILMN_2841290 242 Tnfaip2ILMN_1212695 243 Gpr84ILMN_2623983 244 Egr2ILMN_2474858 245 Tnfaip2ILMN_2991255 246 Gpr84ILMN_1239294 247 Cdc42ep2ILMN_2754985 248 Phlda1ILMN_1250149 249 Dnmt3lILMN_3112268 250 Dnmt3lILMN_1232099 251 Tspan3ILMN_2974720 252 Igf2bp2ILMN_1255973 253 Tnfrsf1bILMN_2593410 254 Kif3cILMN_1215120 255 Sema4aILMN_2760001 256 Ppfibp2ILMN_2881810 257 Gm7609ILMN_2965132 258 Tspyl3ILMN_2755008 259 NfkbizILMN_1251426 260 Klf7ILMN_2964841 261 Ppfibp2ILMN_2594714 262 Ets2ILMN_2772632 263 Saa3ILMN_2419138 264 VclILMN_2790241 265 Herpud1ILMN_2685507 266 Anp32aILMN_2790246 267 Herpud1ILMN_1253972 268 Irak3ILMN_2759079 269 Plpp1ILMN_2763245 270 Cxcl1ILMN_1225085 271 PrkcbILMN_2948143 272 Slc7a11ILMN_2736783 273 Kctd12ILMN_2646166 274 Ndrg1ILMN_2847115 275 Hcar2ILMN_3067259 276 Ppfia3ILMN_2997494 277 LoxILMN_2711075 278 Mmp9ILMN_1229922 279 MarcoILMN_3118707 280 SlaILMN_2722579 281 Slc13a3ILMN_2918312 282 Slc13a3

global

0.8072 50 100

Basal

Myd88

Trif

LPS

Dual

PROBE_IDOrder.log2sa.clustering.then.manualSymbolILMN_1227018 1 Il1aILMN_3160750 2 AA467197ILMN_2630045 3 Il27ILMN_3072427 4 Il1rnILMN_2965903 5 HdcILMN_1245710 6 Ccl2ILMN_1238505 7 Il10ILMN_1249336 8 Mmp13ILMN_2959291 9 Upp1ILMN_1244866 10 Gbp5ILMN_1223499 11 Nos2ILMN_2737713 12 Edn1ILMN_2835117 13 Ccl7ILMN_1242406 14 Dusp2ILMN_1252202 15 Tnfaip3ILMN_2502136 16 Cd40ILMN_1243601 17 Il6ILMN_2790382 18 Gfi1ILMN_2959292 19 Upp1ILMN_2959293 20 Upp1ILMN_2642349 21 Slc7a2ILMN_1253874 22 Serpinb2ILMN_2712075 23 Lcn2ILMN_2668510 24 HpILMN_2668509 25 HpILMN_2944824 26 HpILMN_2616028 27 PtgesILMN_2679276 28 Il19ILMN_2721571 29 Slamf1ILMN_1219915 30 Dgat2ILMN_2966386 31 Rcl1ILMN_1254929 32 Rcl1ILMN_1244424 33 Adora2bILMN_2654754 34 PdpnILMN_2710905 35 S100a8ILMN_1222009 36 Gab1ILMN_2763679 37 Sipa1l1ILMN_3096144 38 Stat3ILMN_2854004 39 SpicILMN_3054914 40 Usp18ILMN_2771766 41 Ccl12ILMN_2595732 42 Nfil3ILMN_3161805 43 Ccl12ILMN_2846776 44 Cdkn1aILMN_2449157 45 Tnfsf9ILMN_1215209 46 Clec4eILMN_2714031 47 Errfi1ILMN_1215862 48 Cxcl9ILMN_2718330 49 CishILMN_2636403 50 Csrnp1ILMN_2811154 51 Cxcl2ILMN_2733778 52 Il4i1ILMN_2752624 53 Ccrl2ILMN_1219869 54 Il15ILMN_2702303 55 Ch25hILMN_1244513 56 Gbp3ILMN_2778655 57 Vcam1ILMN_1231600 58 Ptgs2ILMN_3112011 59 Dusp16ILMN_2754148 60 Dusp16ILMN_2630605 61 Fscn1ILMN_3009243 62 Dusp2ILMN_2638491 63 Slamf7ILMN_2628822 64 Oas1gILMN_2618176 65 Socs3ILMN_2737685 66 Mmp13ILMN_2722380 67 Oasl1ILMN_2777498 68 Il1bILMN_2843286 69 Slc28a2ILMN_1233733 70 Hsp90ab1ILMN_2727920 71 TspoILMN_2727921 72 TspoILMN_1215632 73 Marcksl1ILMN_2617162 74 Marcksl1ILMN_2622671 75 Acsl1ILMN_3083163 76 CpILMN_1235735 77 Slco3a1ILMN_1245200 78 Ifi205ILMN_2707870 79 Mx1ILMN_2731989 80 Ext1ILMN_2918977 81 March1ILMN_2716602 82 Lgals9ILMN_2662926 83 Egr1ILMN_2786442 84 PtgesILMN_2646322 85 Samsn1ILMN_2676066 86 Stard7ILMN_2652608 87 SpicILMN_2622983 88 Dusp1ILMN_1228657 89 Fcgr2bILMN_2422743 90 Tor3aILMN_3102376 91 Fcgr2bILMN_2709820 92 Dtx2ILMN_2701891 93 Marcksl1ILMN_2486186 94 Tnfrsf1bILMN_1256257 95 Isg15ILMN_1236517 96 Il18ILMN_1239770 97 Peli1ILMN_2674979 98 FusILMN_2899863 99 TnfILMN_2503129 100 Cd40ILMN_1230586 101 Traf1ILMN_1230157 102 Rnd3ILMN_2662990 103 Xkr8ILMN_1237695 104 PfkpILMN_2900653 105 Gadd45bILMN_1219074 106 Slfn2ILMN_2590638 107 Bcl2l11ILMN_2480682 108 Sgms1ILMN_2907322 109 Dtx2ILMN_3161253 110 Shisa3ILMN_2717667 111 Pim3ILMN_1246650 112 Hsp90ab1ILMN_2659108 113 Ppp4r1ILMN_1213167 114 Eif4g2ILMN_1230152 115 Adamts4ILMN_2722938 116 Ppp1r15aILMN_2865016 117 Cd83ILMN_1253919 118 Ccl3ILMN_3107690 119 Fam177aILMN_2588051 120 Tubb2aILMN_3115796 121 Cd40ILMN_1230143 122 Batf2ILMN_2742861 123 Serpina3fILMN_2873793 124 Mapkbp1ILMN_2634970 125 Ripk2ILMN_2918002 126 Gbp3ILMN_3115162 127 Jak2ILMN_1216972 128 Clec4eILMN_2680076 129 Ifi205ILMN_2857947 130 Trim36ILMN_2678580 131 Cers6ILMN_2722732 132 SrgnILMN_1230853 133 CfbILMN_2856095 134 Zc3h12aILMN_1216818 135 Malt1ILMN_1255648 136 Csf3rILMN_2725188 137 Atp10aILMN_2606072 138 Agfg1ILMN_2976191 139 Stat5aILMN_2789125 140 Stard7ILMN_3034034 141 Fam177aILMN_2852034 142 Hif1aILMN_1249498 143 PlekILMN_2761900 144 BatfILMN_1221048 145 Psat1ILMN_2947568 146 Gadd45aILMN_2649067 147 Irf1ILMN_3155492 148 TapbpILMN_2674475 149 Tarm1ILMN_2704637 150 MdficILMN_2717127 151 Ddx58ILMN_2752873 152 Lgals9ILMN_2649068 153 Irf1ILMN_2705119 154 Btg1ILMN_1238752 155 NfkbieILMN_2905866 156 Cited2ILMN_1224472 157 Ccl4ILMN_2700166 158 Ccnd2ILMN_2589790 159 Stat3ILMN_3103896 160 Timp1ILMN_2769918 161 Timp1ILMN_2751527 162 SrgnILMN_2926198 163 HilpdaILMN_2662802 164 Ptx3ILMN_2676127 165 Scarf2ILMN_1248181 166 Zbtb7aILMN_2698046 167 Stat3ILMN_2648548 168 Casp7ILMN_2600426 169 Ccdc94ILMN_2868220 170 InhbaILMN_2688912 171 F10ILMN_2706462 172 Il12aILMN_2596596 173 Rgs1ILMN_2596970 174 Tmem39aILMN_1246194 175 Ifit3bILMN_1230458 176 Ifi t3ILMN_2981167 177 Ifi t2ILMN_2846865 178 ActbILMN_2588055 179 ActbILMN_1233172 180 Pou3f1ILMN_2981169 181 Ifi t2ILMN_2696491 182 Phf11aILMN_2903945 183 Gadd45gILMN_2793464 184 Oasl1ILMN_1240864 185 Cmpk2ILMN_2684848 186 Phf11dILMN_2696492 187 Phf11aILMN_2717366 188 Oasl1ILMN_2827780 189 Phf11dILMN_2711910 190 Ifnb1ILMN_3142803 191 Cxcl10ILMN_1227573 192 Irf7ILMN_2923615 193 Ppm1kILMN_2944666 194 Ifi t3ILMN_3122961 195 Gbp2ILMN_1230357 196 Itpr1ILMN_1237736 197 Plekhf2ILMN_2845658 198 BC147527ILMN_2743244 199 Cd86ILMN_2744890 200 Gadd45gILMN_1234539 201 Irgm1ILMN_2492716 202 UacaILMN_1259564 203 Irgm2ILMN_1244891 204 Cst7ILMN_1217041 205 Cd86ILMN_1225204 206 Rsad2ILMN_2598622 207 Slamf8ILMN_2652857 208 Ifi47ILMN_2762380 209 Enpp4ILMN_2959253 210 Dnaaf3ILMN_2626939 211 DaxxILMN_2870302 212 Parp3ILMN_2860645 213 Gbp7ILMN_1213978 214 PtproILMN_2829594 215 Hspa1aILMN_1249864 216 Fam26fILMN_2603081 217 Nt5c3ILMN_1239219 218 Mx2ILMN_2725927 219 Serpina3gILMN_2599782 220 Irf1ILMN_2654554 221 Fgl2ILMN_1247710 222 Nod1ILMN_2971721 223 Fbrsl1ILMN_2610234 224 Il15ILMN_2834777 225 Irf1ILMN_1218240 226 Cd69ILMN_2990616 227 Birc2ILMN_2938704 228 Il15ILMN_2902979 229 FasILMN_1250438 230 Marcksl1ILMN_2897891 231 Rgs1ILMN_2618918 232 Slc2a6ILMN_2804166 233 Igsf9ILMN_2690551 234 NfkbieILMN_3148543 235 SrcILMN_2625377 236 Rgs1ILMN_2663374 237 Nr1h3ILMN_1221750 238 MyclILMN_1246609 239 Rasgrp1ILMN_2728729 240 Sdc4ILMN_2628757 241 Ehd1ILMN_2841290 242 Tnfaip2ILMN_1212695 243 Gpr84ILMN_2623983 244 Egr2ILMN_2474858 245 Tnfaip2ILMN_2991255 246 Gpr84ILMN_1239294 247 Cdc42ep2ILMN_2754985 248 Phlda1ILMN_1250149 249 Dnmt3lILMN_3112268 250 Dnmt3lILMN_1232099 251 Tspan3ILMN_2974720 252 Igf2bp2ILMN_1255973 253 Tnfrsf1bILMN_2593410 254 Kif3cILMN_1215120 255 Sema4aILMN_2760001 256 Ppfibp2ILMN_2881810 257 Gm7609ILMN_2965132 258 Tspyl3ILMN_2755008 259 NfkbizILMN_1251426 260 Klf7ILMN_2964841 261 Ppfibp2ILMN_2594714 262 Ets2ILMN_2772632 263 Saa3ILMN_2419138 264 VclILMN_2790241 265 Herpud1ILMN_2685507 266 Anp32aILMN_2790246 267 Herpud1ILMN_1253972 268 Irak3ILMN_2759079 269 Plpp1ILMN_2763245 270 Cxcl1ILMN_1225085 271 PrkcbILMN_2948143 272 Slc7a11ILMN_2736783 273 Kctd12ILMN_2646166 274 Ndrg1ILMN_2847115 275 Hcar2ILMN_3067259 276 Ppfia3ILMN_2997494 277 LoxILMN_2711075 278 Mmp9ILMN_1229922 279 MarcoILMN_3118707 280 SlaILMN_2722579 281 Slc13a3ILMN_2918312 282 Slc13a3

DIF values (log2) % expressionglobal

-5 .09 0 6.07

RI_1h

RI_4h

RI_8h

IP_1h

IP_4h

IP_8h

RL_1h

RL_4h

RL_8h

PL_1h

PL_4h

PL_8h

IL_1h

IL_4h

IL_8h

RP_1h

RP_4h

RP_8h

SymbolIl1aAA467197Il27Il1rnHdcCcl2Il10Mmp13Upp1Gbp5Nos2Edn1Ccl7Dusp2Tnfaip3Cd40Il6Gfi1Upp1Upp1Slc7a2Serpinb2Lcn2HpHpHpPtgesIl19Slamf1Dgat2Rcl1Rcl1Adora2bPdpnS100a8Gab1Sipa1l1Stat3SpicUsp18Ccl12Nfil3Ccl12Cdkn1aTnfsf9Clec4eErrfi1Cxcl9CishCsrnp1Cxcl2Il4i1Ccrl2Il15Ch25hGbp3Vcam1Ptgs2Dusp2Slamf7Oas1gSocs3Mmp13Oasl1Il1bSlc28a2Hsp90ab1TspoTspoMarcksl1Marcksl1Acsl1Dusp16Dusp16Fscn1CpSlco3a1Ifi205Mx1Ext1March1Lgals9Egr1PtgesSamsn1Stard7SpicDusp1Fcgr2bTor3aFcgr2bDtx2Marcksl1Tnfrsf1bIsg15Il18Peli1FusTnfCd40Traf1Rnd3Xkr8PfkpGadd45bSlfn2Bcl2l11Sgms1Dtx2Shisa3Pim3Hsp90ab1Ppp4r1Eif4g2Adamts4Ppp1r15aCd83Ccl3Fam177aTubb2aCd40Batf2Serpina3fMapkbp1Ripk2Gbp3Jak2Clec4eIfi205Trim36Cers6SrgnCfbZc3h12aMalt1Csf3rAtp10aAgfg1Stat5aStard7Fam177aHif1aPlekBatfPsat1Gadd45aIrf1TapbpTarm1MdficDdx58Lgals9Irf1Btg1NfkbieCited2Ccl4Ccnd2Stat3Timp1Timp1SrgnHilpdaPtx3Scarf2Zbtb7aStat3Casp7Ccdc94InhbaF10Il12aRgs1Tmem39aAnp32aSrcMarcksl1Tnfrsf1bTspan3Egr2Cd69Il15Il15Mx2Cd86Parp3Fbrsl1Cd86FasSlamf8Ifi47Birc2Cst7Phf11aIrgm1Irf7Fgl2Enpp4Phf11aPhf11dIfi t2Irgm2Dnaaf3Rsad2Phf11dIfi t3Nod1Sema4aIfnb1MyclDaxxOasl1Oasl1Hspa1aRgs1PtproIrf1NfkbieRgs1ActbActbBC147527Itpr1Nt5c3Gbp2Irf1If i t2Cxcl10Gm7609Sdc4Hcar2Fam26fGbp7Cmpk2Slc2a6Klf7Plekhf2Ppm1kIfit3bIf i t3Gadd45gGadd45gUacaPou3f1Saa3Cxcl1Ehd1NfkbizVclTnfaip2Ndrg1Rasgrp1Tnfaip2Gpr84Nr1h3Plpp1Phlda1Serpina3gPrkcbMmp9MarcoKctd12SlaLoxSlc13a3Slc13a3Dnmt3lIgsf9Dnmt3lIrak3Gpr84Slc7a11Ppfia3Cdc42ep2Tspyl3Kif3cPpfibp2Igf2bp2Ets2Ppfibp2Herpud1Herpud1

global

0.3283 50 100

Basal

Myd88

Trif

Synergy

WT

SymbolIl6Slc7a2Il1aEdn1Serpinb2Il1bIl10NfkbizCxcl1Errfi1Peli1Ifnb1

-5 0 6 0 50 100

Basa

lM

yD88

TRIF

LPS

Dua

l

global

-2 .21 2.37 6.95

R848_1h

R848_4h

R848_8h

pIC_1h

pIC_4h

pIC_8h

Pam_1h

Pam_4h

Pam_8h

LPS_1h

LPS_4h

LPS_8h

RI_1h

RI_4h

RI_8h

IP_1h

IP_4h

IP_8h

RL_1h

RL_4h

RL_8h

PL_1h

PL_4h

PL_8h

IL_1h

IL_4h

IL_8h

RP_1h

RP_4h

RP_8h

PROBE_IDOrder.log2sa.clustering.then.manualSymbolILMN_1227018 1 Il1aILMN_3160750 2 AA467197ILMN_2630045 3 Il27ILMN_3072427 4 Il1rnILMN_2965903 5 HdcILMN_1245710 6 Ccl2ILMN_1238505 7 Il10ILMN_1249336 8 Mmp13ILMN_2959291 9 Upp1ILMN_1244866 10 Gbp5ILMN_1223499 11 Nos2ILMN_2737713 12 Edn1ILMN_2835117 13 Ccl7ILMN_1242406 14 Dusp2ILMN_1252202 15 Tnfaip3ILMN_2502136 16 Cd40ILMN_1243601 17 Il6ILMN_2790382 18 Gfi1ILMN_2959292 19 Upp1ILMN_2959293 20 Upp1ILMN_2642349 21 Slc7a2ILMN_1253874 22 Serpinb2ILMN_2712075 23 Lcn2ILMN_2668510 24 HpILMN_2668509 25 HpILMN_2944824 26 HpILMN_2616028 27 PtgesILMN_2679276 28 Il19ILMN_2721571 29 Slamf1ILMN_1219915 30 Dgat2ILMN_2966386 31 Rcl1ILMN_1254929 32 Rcl1ILMN_1244424 33 Adora2bILMN_2654754 34 PdpnILMN_2710905 35 S100a8ILMN_1222009 36 Gab1ILMN_2763679 37 Sipa1l1ILMN_3096144 38 Stat3ILMN_2854004 39 SpicILMN_3054914 40 Usp18ILMN_2771766 41 Ccl12ILMN_2595732 42 Nfil3ILMN_3161805 43 Ccl12ILMN_2846776 44 Cdkn1aILMN_2449157 45 Tnfsf9ILMN_1215209 46 Clec4eILMN_2714031 47 Errfi1ILMN_1215862 48 Cxcl9ILMN_2718330 49 CishILMN_2636403 50 Csrnp1ILMN_2811154 51 Cxcl2ILMN_2733778 52 Il4i1ILMN_2752624 53 Ccrl2ILMN_1219869 54 Il15ILMN_2702303 55 Ch25hILMN_1244513 56 Gbp3ILMN_2778655 57 Vcam1ILMN_1231600 58 Ptgs2ILMN_3112011 59 Dusp16ILMN_2754148 60 Dusp16ILMN_2630605 61 Fscn1ILMN_3009243 62 Dusp2ILMN_2638491 63 Slamf7ILMN_2628822 64 Oas1gILMN_2618176 65 Socs3ILMN_2737685 66 Mmp13ILMN_2722380 67 Oasl1ILMN_2777498 68 Il1bILMN_2843286 69 Slc28a2ILMN_1233733 70 Hsp90ab1ILMN_2727920 71 TspoILMN_2727921 72 TspoILMN_1215632 73 Marcksl1ILMN_2617162 74 Marcksl1ILMN_2622671 75 Acsl1ILMN_3083163 76 CpILMN_1235735 77 Slco3a1ILMN_1245200 78 Ifi205ILMN_2707870 79 Mx1ILMN_2731989 80 Ext1ILMN_2918977 81 March1ILMN_2716602 82 Lgals9ILMN_2662926 83 Egr1ILMN_2786442 84 PtgesILMN_2646322 85 Samsn1ILMN_2676066 86 Stard7ILMN_2652608 87 SpicILMN_2622983 88 Dusp1ILMN_1228657 89 Fcgr2bILMN_2422743 90 Tor3aILMN_3102376 91 Fcgr2bILMN_2709820 92 Dtx2ILMN_2701891 93 Marcksl1ILMN_2486186 94 Tnfrsf1bILMN_1256257 95 Isg15ILMN_1236517 96 Il18ILMN_1239770 97 Peli1ILMN_2674979 98 FusILMN_2899863 99 TnfILMN_2503129 100 Cd40ILMN_1230586 101 Traf1ILMN_1230157 102 Rnd3ILMN_2662990 103 Xkr8ILMN_1237695 104 PfkpILMN_2900653 105 Gadd45bILMN_1219074 106 Slfn2ILMN_2590638 107 Bcl2l11ILMN_2480682 108 Sgms1ILMN_2907322 109 Dtx2ILMN_3161253 110 Shisa3ILMN_2717667 111 Pim3ILMN_1246650 112 Hsp90ab1ILMN_2659108 113 Ppp4r1ILMN_1213167 114 Eif4g2ILMN_1230152 115 Adamts4ILMN_2722938 116 Ppp1r15aILMN_2865016 117 Cd83ILMN_1253919 118 Ccl3ILMN_3107690 119 Fam177aILMN_2588051 120 Tubb2aILMN_3115796 121 Cd40ILMN_1230143 122 Batf2ILMN_2742861 123 Serpina3fILMN_2873793 124 Mapkbp1ILMN_2634970 125 Ripk2ILMN_2918002 126 Gbp3ILMN_3115162 127 Jak2ILMN_1216972 128 Clec4eILMN_2680076 129 Ifi205ILMN_2857947 130 Trim36ILMN_2678580 131 Cers6ILMN_2722732 132 SrgnILMN_1230853 133 CfbILMN_2856095 134 Zc3h12aILMN_1216818 135 Malt1ILMN_1255648 136 Csf3rILMN_2725188 137 Atp10aILMN_2606072 138 Agfg1ILMN_2976191 139 Stat5aILMN_2789125 140 Stard7ILMN_3034034 141 Fam177aILMN_2852034 142 Hif1aILMN_1249498 143 PlekILMN_2761900 144 BatfILMN_1221048 145 Psat1ILMN_2947568 146 Gadd45aILMN_2649067 147 Irf1ILMN_3155492 148 TapbpILMN_2674475 149 Tarm1ILMN_2704637 150 MdficILMN_2717127 151 Ddx58ILMN_2752873 152 Lgals9ILMN_2649068 153 Irf1ILMN_2705119 154 Btg1ILMN_1238752 155 NfkbieILMN_2905866 156 Cited2ILMN_1224472 157 Ccl4ILMN_2700166 158 Ccnd2ILMN_2589790 159 Stat3ILMN_3103896 160 Timp1ILMN_2769918 161 Timp1ILMN_2751527 162 SrgnILMN_2926198 163 HilpdaILMN_2662802 164 Ptx3ILMN_2676127 165 Scarf2ILMN_1248181 166 Zbtb7aILMN_2698046 167 Stat3ILMN_2648548 168 Casp7ILMN_2600426 169 Ccdc94ILMN_2868220 170 InhbaILMN_2688912 171 F10ILMN_2706462 172 Il12aILMN_2596596 173 Rgs1ILMN_2596970 174 Tmem39aILMN_1246194 175 Ifit3bILMN_1230458 176 Ifi t3ILMN_2981167 177 Ifi t2ILMN_2846865 178 ActbILMN_2588055 179 ActbILMN_1233172 180 Pou3f1ILMN_2981169 181 Ifi t2ILMN_2696491 182 Phf11aILMN_2903945 183 Gadd45gILMN_2793464 184 Oasl1ILMN_1240864 185 Cmpk2ILMN_2684848 186 Phf11dILMN_2696492 187 Phf11aILMN_2717366 188 Oasl1ILMN_2827780 189 Phf11dILMN_2711910 190 Ifnb1ILMN_3142803 191 Cxcl10ILMN_1227573 192 Irf7ILMN_2923615 193 Ppm1kILMN_2944666 194 Ifi t3ILMN_3122961 195 Gbp2ILMN_1230357 196 Itpr1ILMN_1237736 197 Plekhf2ILMN_2845658 198 BC147527ILMN_2743244 199 Cd86ILMN_2744890 200 Gadd45gILMN_1234539 201 Irgm1ILMN_2492716 202 UacaILMN_1259564 203 Irgm2ILMN_1244891 204 Cst7ILMN_1217041 205 Cd86ILMN_1225204 206 Rsad2ILMN_2598622 207 Slamf8ILMN_2652857 208 Ifi47ILMN_2762380 209 Enpp4ILMN_2959253 210 Dnaaf3ILMN_2626939 211 DaxxILMN_2870302 212 Parp3ILMN_2860645 213 Gbp7ILMN_1213978 214 PtproILMN_2829594 215 Hspa1aILMN_1249864 216 Fam26fILMN_2603081 217 Nt5c3ILMN_1239219 218 Mx2ILMN_2725927 219 Serpina3gILMN_2599782 220 Irf1ILMN_2654554 221 Fgl2ILMN_1247710 222 Nod1ILMN_2971721 223 Fbrsl1ILMN_2610234 224 Il15ILMN_2834777 225 Irf1ILMN_1218240 226 Cd69ILMN_2990616 227 Birc2ILMN_2938704 228 Il15ILMN_2902979 229 FasILMN_1250438 230 Marcksl1ILMN_2897891 231 Rgs1ILMN_2618918 232 Slc2a6ILMN_2804166 233 Igsf9ILMN_2690551 234 NfkbieILMN_3148543 235 SrcILMN_2625377 236 Rgs1ILMN_2663374 237 Nr1h3ILMN_1221750 238 MyclILMN_1246609 239 Rasgrp1ILMN_2728729 240 Sdc4ILMN_2628757 241 Ehd1ILMN_2841290 242 Tnfaip2ILMN_1212695 243 Gpr84ILMN_2623983 244 Egr2ILMN_2474858 245 Tnfaip2ILMN_2991255 246 Gpr84ILMN_1239294 247 Cdc42ep2ILMN_2754985 248 Phlda1ILMN_1250149 249 Dnmt3lILMN_3112268 250 Dnmt3lILMN_1232099 251 Tspan3ILMN_2974720 252 Igf2bp2ILMN_1255973 253 Tnfrsf1bILMN_2593410 254 Kif3cILMN_1215120 255 Sema4aILMN_2760001 256 Ppfibp2ILMN_2881810 257 Gm7609ILMN_2965132 258 Tspyl3ILMN_2755008 259 NfkbizILMN_1251426 260 Klf7ILMN_2964841 261 Ppfibp2ILMN_2594714 262 Ets2ILMN_2772632 263 Saa3ILMN_2419138 264 VclILMN_2790241 265 Herpud1ILMN_2685507 266 Anp32aILMN_2790246 267 Herpud1ILMN_1253972 268 Irak3ILMN_2759079 269 Plpp1ILMN_2763245 270 Cxcl1ILMN_1225085 271 PrkcbILMN_2948143 272 Slc7a11ILMN_2736783 273 Kctd12ILMN_2646166 274 Ndrg1ILMN_2847115 275 Hcar2ILMN_3067259 276 Ppfia3ILMN_2997494 277 LoxILMN_2711075 278 Mmp9ILMN_1229922 279 MarcoILMN_3118707 280 SlaILMN_2722579 281 Slc13a3ILMN_2918312 282 Slc13a3

Normalized mRNA level (log2)

-2 0 6

global

-2 .21 2.37 6.95

R848_1h

R848_4h

R848_8h

pIC_1h

pIC_4h

pIC_8h

Pam_1h

Pam_4h

Pam_8h

LPS_1h

LPS_4h

LPS_8h

RI_1h

RI_4h

RI_8h

IP_1h

IP_4h

IP_8h

RL_1h

RL_4h

RL_8h

PL_1h

PL_4h

PL_8h

IL_1h

IL_4h

IL_8h

RP_1h

RP_4h

RP_8h

PROBE_IDOrder.log2sa.clustering.then.manualSymbolILMN_1227018 1 Il1aILMN_3160750 2 AA467197ILMN_2630045 3 Il27ILMN_3072427 4 Il1rnILMN_2965903 5 HdcILMN_1245710 6 Ccl2ILMN_1238505 7 Il10ILMN_1249336 8 Mmp13ILMN_2959291 9 Upp1ILMN_1244866 10 Gbp5ILMN_1223499 11 Nos2ILMN_2737713 12 Edn1ILMN_2835117 13 Ccl7ILMN_1242406 14 Dusp2ILMN_1252202 15 Tnfaip3ILMN_2502136 16 Cd40ILMN_1243601 17 Il6ILMN_2790382 18 Gfi1ILMN_2959292 19 Upp1ILMN_2959293 20 Upp1ILMN_2642349 21 Slc7a2ILMN_1253874 22 Serpinb2ILMN_2712075 23 Lcn2ILMN_2668510 24 HpILMN_2668509 25 HpILMN_2944824 26 HpILMN_2616028 27 PtgesILMN_2679276 28 Il19ILMN_2721571 29 Slamf1ILMN_1219915 30 Dgat2ILMN_2966386 31 Rcl1ILMN_1254929 32 Rcl1ILMN_1244424 33 Adora2bILMN_2654754 34 PdpnILMN_2710905 35 S100a8ILMN_1222009 36 Gab1ILMN_2763679 37 Sipa1l1ILMN_3096144 38 Stat3ILMN_2854004 39 SpicILMN_3054914 40 Usp18ILMN_2771766 41 Ccl12ILMN_2595732 42 Nfil3ILMN_3161805 43 Ccl12ILMN_2846776 44 Cdkn1aILMN_2449157 45 Tnfsf9ILMN_1215209 46 Clec4eILMN_2714031 47 Errfi1ILMN_1215862 48 Cxcl9ILMN_2718330 49 CishILMN_2636403 50 Csrnp1ILMN_2811154 51 Cxcl2ILMN_2733778 52 Il4i1ILMN_2752624 53 Ccrl2ILMN_1219869 54 Il15ILMN_2702303 55 Ch25hILMN_1244513 56 Gbp3ILMN_2778655 57 Vcam1ILMN_1231600 58 Ptgs2ILMN_3112011 59 Dusp16ILMN_2754148 60 Dusp16ILMN_2630605 61 Fscn1ILMN_3009243 62 Dusp2ILMN_2638491 63 Slamf7ILMN_2628822 64 Oas1gILMN_2618176 65 Socs3ILMN_2737685 66 Mmp13ILMN_2722380 67 Oasl1ILMN_2777498 68 Il1bILMN_2843286 69 Slc28a2ILMN_1233733 70 Hsp90ab1ILMN_2727920 71 TspoILMN_2727921 72 TspoILMN_1215632 73 Marcksl1ILMN_2617162 74 Marcksl1ILMN_2622671 75 Acsl1ILMN_3083163 76 CpILMN_1235735 77 Slco3a1ILMN_1245200 78 Ifi205ILMN_2707870 79 Mx1ILMN_2731989 80 Ext1ILMN_2918977 81 March1ILMN_2716602 82 Lgals9ILMN_2662926 83 Egr1ILMN_2786442 84 PtgesILMN_2646322 85 Samsn1ILMN_2676066 86 Stard7ILMN_2652608 87 SpicILMN_2622983 88 Dusp1ILMN_1228657 89 Fcgr2bILMN_2422743 90 Tor3aILMN_3102376 91 Fcgr2bILMN_2709820 92 Dtx2ILMN_2701891 93 Marcksl1ILMN_2486186 94 Tnfrsf1bILMN_1256257 95 Isg15ILMN_1236517 96 Il18ILMN_1239770 97 Peli1ILMN_2674979 98 FusILMN_2899863 99 TnfILMN_2503129 100 Cd40ILMN_1230586 101 Traf1ILMN_1230157 102 Rnd3ILMN_2662990 103 Xkr8ILMN_1237695 104 PfkpILMN_2900653 105 Gadd45bILMN_1219074 106 Slfn2ILMN_2590638 107 Bcl2l11ILMN_2480682 108 Sgms1ILMN_2907322 109 Dtx2ILMN_3161253 110 Shisa3ILMN_2717667 111 Pim3ILMN_1246650 112 Hsp90ab1ILMN_2659108 113 Ppp4r1ILMN_1213167 114 Eif4g2ILMN_1230152 115 Adamts4ILMN_2722938 116 Ppp1r15aILMN_2865016 117 Cd83ILMN_1253919 118 Ccl3ILMN_3107690 119 Fam177aILMN_2588051 120 Tubb2aILMN_3115796 121 Cd40ILMN_1230143 122 Batf2ILMN_2742861 123 Serpina3fILMN_2873793 124 Mapkbp1ILMN_2634970 125 Ripk2ILMN_2918002 126 Gbp3ILMN_3115162 127 Jak2ILMN_1216972 128 Clec4eILMN_2680076 129 Ifi205ILMN_2857947 130 Trim36ILMN_2678580 131 Cers6ILMN_2722732 132 SrgnILMN_1230853 133 CfbILMN_2856095 134 Zc3h12aILMN_1216818 135 Malt1ILMN_1255648 136 Csf3rILMN_2725188 137 Atp10aILMN_2606072 138 Agfg1ILMN_2976191 139 Stat5aILMN_2789125 140 Stard7ILMN_3034034 141 Fam177aILMN_2852034 142 Hif1aILMN_1249498 143 PlekILMN_2761900 144 BatfILMN_1221048 145 Psat1ILMN_2947568 146 Gadd45aILMN_2649067 147 Irf1ILMN_3155492 148 TapbpILMN_2674475 149 Tarm1ILMN_2704637 150 MdficILMN_2717127 151 Ddx58ILMN_2752873 152 Lgals9ILMN_2649068 153 Irf1ILMN_2705119 154 Btg1ILMN_1238752 155 NfkbieILMN_2905866 156 Cited2ILMN_1224472 157 Ccl4ILMN_2700166 158 Ccnd2ILMN_2589790 159 Stat3ILMN_3103896 160 Timp1ILMN_2769918 161 Timp1ILMN_2751527 162 SrgnILMN_2926198 163 HilpdaILMN_2662802 164 Ptx3ILMN_2676127 165 Scarf2ILMN_1248181 166 Zbtb7aILMN_2698046 167 Stat3ILMN_2648548 168 Casp7ILMN_2600426 169 Ccdc94ILMN_2868220 170 InhbaILMN_2688912 171 F10ILMN_2706462 172 Il12aILMN_2596596 173 Rgs1ILMN_2596970 174 Tmem39aILMN_1246194 175 Ifit3bILMN_1230458 176 Ifi t3ILMN_2981167 177 Ifi t2ILMN_2846865 178 ActbILMN_2588055 179 ActbILMN_1233172 180 Pou3f1ILMN_2981169 181 Ifi t2ILMN_2696491 182 Phf11aILMN_2903945 183 Gadd45gILMN_2793464 184 Oasl1ILMN_1240864 185 Cmpk2ILMN_2684848 186 Phf11dILMN_2696492 187 Phf11aILMN_2717366 188 Oasl1ILMN_2827780 189 Phf11dILMN_2711910 190 Ifnb1ILMN_3142803 191 Cxcl10ILMN_1227573 192 Irf7ILMN_2923615 193 Ppm1kILMN_2944666 194 Ifi t3ILMN_3122961 195 Gbp2ILMN_1230357 196 Itpr1ILMN_1237736 197 Plekhf2ILMN_2845658 198 BC147527ILMN_2743244 199 Cd86ILMN_2744890 200 Gadd45gILMN_1234539 201 Irgm1ILMN_2492716 202 UacaILMN_1259564 203 Irgm2ILMN_1244891 204 Cst7ILMN_1217041 205 Cd86ILMN_1225204 206 Rsad2ILMN_2598622 207 Slamf8ILMN_2652857 208 Ifi47ILMN_2762380 209 Enpp4ILMN_2959253 210 Dnaaf3ILMN_2626939 211 DaxxILMN_2870302 212 Parp3ILMN_2860645 213 Gbp7ILMN_1213978 214 PtproILMN_2829594 215 Hspa1aILMN_1249864 216 Fam26fILMN_2603081 217 Nt5c3ILMN_1239219 218 Mx2ILMN_2725927 219 Serpina3gILMN_2599782 220 Irf1ILMN_2654554 221 Fgl2ILMN_1247710 222 Nod1ILMN_2971721 223 Fbrsl1ILMN_2610234 224 Il15ILMN_2834777 225 Irf1ILMN_1218240 226 Cd69ILMN_2990616 227 Birc2ILMN_2938704 228 Il15ILMN_2902979 229 FasILMN_1250438 230 Marcksl1ILMN_2897891 231 Rgs1ILMN_2618918 232 Slc2a6ILMN_2804166 233 Igsf9ILMN_2690551 234 NfkbieILMN_3148543 235 SrcILMN_2625377 236 Rgs1ILMN_2663374 237 Nr1h3ILMN_1221750 238 MyclILMN_1246609 239 Rasgrp1ILMN_2728729 240 Sdc4ILMN_2628757 241 Ehd1ILMN_2841290 242 Tnfaip2ILMN_1212695 243 Gpr84ILMN_2623983 244 Egr2ILMN_2474858 245 Tnfaip2ILMN_2991255 246 Gpr84ILMN_1239294 247 Cdc42ep2ILMN_2754985 248 Phlda1ILMN_1250149 249 Dnmt3lILMN_3112268 250 Dnmt3lILMN_1232099 251 Tspan3ILMN_2974720 252 Igf2bp2ILMN_1255973 253 Tnfrsf1bILMN_2593410 254 Kif3cILMN_1215120 255 Sema4aILMN_2760001 256 Ppfibp2ILMN_2881810 257 Gm7609ILMN_2965132 258 Tspyl3ILMN_2755008 259 NfkbizILMN_1251426 260 Klf7ILMN_2964841 261 Ppfibp2ILMN_2594714 262 Ets2ILMN_2772632 263 Saa3ILMN_2419138 264 VclILMN_2790241 265 Herpud1ILMN_2685507 266 Anp32aILMN_2790246 267 Herpud1ILMN_1253972 268 Irak3ILMN_2759079 269 Plpp1ILMN_2763245 270 Cxcl1ILMN_1225085 271 PrkcbILMN_2948143 272 Slc7a11ILMN_2736783 273 Kctd12ILMN_2646166 274 Ndrg1ILMN_2847115 275 Hcar2ILMN_3067259 276 Ppfia3ILMN_2997494 277 LoxILMN_2711075 278 Mmp9ILMN_1229922 279 MarcoILMN_3118707 280 SlaILMN_2722579 281 Slc13a3ILMN_2918312 282 Slc13a3

2 4

Figure S4 continued

Right panel: Percent mRNA expression normalized to the maximum level (100%) for each gene, under basalconditions, treatment with a MyD88 ligand alone, a TRIF ligand alone, LPS or dual ligand. For the MyD88alone (either R848 or Pam3CSK4) and dual ligand conditions (6 pairwise PAMP combinations), the highestexpression value is shown. Left panel: Measured mRNA levels (log2) of the described 282 non-additivetranscripts in response to 4 single ligand stimulations and 6 pair wise dual ligand stimulations for 1, 4, and 8 hr.Data were derived from duplicate microarrays (see Methods; Data files S1 and S2).

a

b

3418 113

16

Lcn2, Il19, Hp, Il12a, Il6

Slamf1, Dgat2, Nos2, Nfil3

Il1a, Il1b, Il1rn, Mmp13, AA467197, Socs3,Upp1, Edn1, Il27, Cxcl2, Ccl12, Il10, Il15, Cd40

LPS induced

Ifnb1, Ifit3, Mx1, Oasl1, Ddx58, Irf1, Stat3

TRIF induced MyD88 induced

Common

Functional Class

Gene Synergy (max DIF)

Il1a 60.45 Il27 22.23

Cxcl2 20.9 Il19 19.29

Ccl12 16.98 Il1b 14.26 Il10 12.51

Ccl12 11.63 Ccrl2 10.53 Cxcl9 10.12 Ccl7 9.92

Cytokine

Functional Class

Gene Synergy (max DIF)

Lcn2 67.33 Mmp13 33.6 Edn1 22.53

Mmp13 19.4 Hdc 14.73 Cd40 13.85 Gbp5 11.07 Ptges 10.81 Spic 9.81

Effector

Functional Class

Gene Synergy (max DIF)

AA467197 32.53 Socs3 24.57 Il1rn 24.21

Dusp2 18.56 Dusp2 13.36 Usp18 13.22

Suppressor

Functional Class

Gene Synergy (max DIF)

Upp1 23.18 Upp1 13.64

Slc28a2 10.91 Cp 9.45

Other

Figure S5. Related to Figure 3.Synergized genes are enriched for key immune mediators. a) MyD88- and TRIF-dependency of 181synergized transcripts. Numbers indicate the number of transcripts in respective classes. b) Functional classesof top 30 synergized transcripts showing that synergized genes are enriched for key immune mediators.

Target siRNA + 848 or PIC/848

NTC siRNA + 848

NTC siRNA+ PIC/848

NTC siRNA + PIC

siGFP control

Cells only control

Secondary screen plate map 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

A

B

C

D

F

G

H

I

J

K

L

M

N

O

P

E

1 2 3 1 2 3Replicate Replicate

0.4

0.8

1.2

0.0

Irak2 Hist1h2ap

Positive regulators:

1 2 3 1 2 3Replicate Replicate

2

4

6

0

Tnip1 Tnfaip3

Negative regulators:

IL-6

leve

l (n

orm

aliz

ed)

a

b

Figure S6. Related to Figure 5.Examples of positive and negative regulators and secondary screen plate layout for the RNAi screen.(a) Examples of primary RNAi screen data output for positive (Irak2 and Hist1h2ap) and negative (Tnip1 andTnfaip3) regulators of dual TLR ligand induced IL-6 secretion. (b) Secondary RNAi screen plate map. NTC;Non-targeting control, 848: R848, PIC: poly (I:C).

Figure S7.(a) Pathway analysis of screen hits (Related to Figure 6). Ingenuity Pathway Analysis output for screen hitswith known associations with NF-kB regulation. Screen hits are indicated by grey shading, genes connected tothe hits are white. Arrows indicate a known functional link between the genes.(b) RNAi knockdown efficiency of screen hits in BMDM (Related to Figure 7). RNAi knockdown efficiencyof four selected screen hits (Helz2, Phf11d, Zscan12, and Sertad3) in BMDMs treated with 5 nM Lipid A for 0,1, 2 and 8 hr. Two independent siRNAs were used for each hit gene. mRNA expression levels relative toBMDM transfected with NC1#1 non-targeting control siRNA are shown by a color gradient from blue (0%) tored (100%).

a

bglobal

0 5 0 100

0 h

1 h

2 h

8 h

AnnotationNC#1

Helz2_siRNA1

Helz2_siRNA2

Sertad3_siRNA1

Sertad3_siRNA2

Phf11d_siRNA1

Phf11d_siRNA2

Zscan12_siRNA1

Zscan12_siRNA2

global

0 5 0 100

0 h

1 h

2 h

8 h

AnnotationNC#1

Helz2_siRNA1

Helz2_siRNA2

Sertad3_siRNA1

Sertad3_siRNA2

Phf11d_siRNA1

Phf11d_siRNA2

Zscan12_siRNA1

Zscan12_siRNA2

0 1 2 8

Time (hr)

global

0 5 0 100

0 h

1 h

2 h

8 h

AnnotationNC#1

Helz2_siRNA1

Helz2_siRNA2

Sertad3_siRNA1

Sertad3_siRNA2

Phf11d_siRNA1

Phf11d_siRNA2

Zscan12_siRNA1

Zscan12_siRNA2

mRNA level (%)

0 50 100