supporting information - pnas...2009/10/26 · microsoft excel:mac v.x (microsoft corporation)....

Supporting InformationLiu et al. 10.1073/pnas.0910009106SI TextQuantitative Real-Time PCR. Oligonucleotides used for RT-PCRinclude: Human Twist: Forward: 5�- ggagtccgcagtcttacgag, Re-verse: 5�- cctctaccaggtcctccaga; Nanog: Forward: CA-GAAAAACCAGTGGTTGAAGACTAG, Reverse: GCAAT-GGATGCTGGGATACTC (1); Oct4: Forward: CTGTA-GGGAGGGCTTCGGGCACTT, Reverse: CTGAGGGC-CAGGCAGGAGCACGAG (2); Sox2: Forward: GGCAGCTA-CAGCATGATGCAGGAGC, Reverse: CTGGTCATGGAGT-TGTACTGCAGG (2); KLF4: Forward: TGCCAGA-CCAGATGCAGTCAC, Reverse: GTAGTGCCTGGTCAGT-TCATC (3); c-Myc: Forward: TGAGCCCCTAGTGCTGCAT,Reverse: AGCCCGACTCCGACCTCTT; 18s rRNA oligos (4).

Cell Culture, Retroviral Infection, Luciferase Reporter Assay, siRNATransfection, and 3-D Culture. The Twist-specific siRNA targetsequences is GGACAAGCTGAGCAAGATTCA (5, 6). Thep21CIP1 siRNA sequence is GCCTTAGTCTCAGTTTGGT-GTCTT (7). Twist and p21CIP1-specific siRNAs or control siR-NAs (100 nM) were transfected by the Nucleofector technologyusing Nucleofector Kit V and program T-024 (Amaxa Biosystems).Transfection efficiency was monitored by non-silencing fluoresce-in-labeled siRNA from QIAGEN. For 3-D culture, 2,000 cellsmixed with growth factor reduced Matrigel matrix (volume ratio1:1) were seeded into a four-well chamber and photographedaccording to a previously described protocol (8, 9).

Mammosphere Formation and FACS Analysis of Stem Cell SurfaceMarkers. Before labeling, the cells were blocked with normalmouse IgG in 1/100 dilution for 30 min and then incubated withPE labeled mouse anti-human CD24 (1/5) (clone ML5, BDPharMingen) and/or PE/Cy5 labeled rat anti human/mouseCD44 (1/200) (clone IM7, BioLegend) for 1 h. All experimentswere conducted at 4 °C. Cell sorting was performed on aFACSCalibur cell sorter (BD Biosciences). The data wereanalyzed with FlowJo software (Tree Star, Inc.).

Western Blot Analysis, Immunofluorescence, and Immunohistochem-istry (IHC). IHC was following conducted as described in ref. 10.Briefly, after slides were rehydrated in graded alcohol, retrievedfor antigen using Citra (Biogenex) in a steamer, and blocked withperoxidase and goat serum, the slides were incubated with anantibody specific for E-cadherin (#610182, BD Biosciences),vimentin (M7020, Dako), or Twist (T6451, Sigma); all dilutionswere 1:50. For the rabbit primary antibodies, slides were incu-bated with Envision System labeled polymer-HRP anti-rabbitsecondary antibody (K4003, Dako) with TSA-plus Cyanine 5system (NEL745001KT, Perkin-Elmer LAS). For the mouseprimary antibodies, slides were stained using the M.O.M. basickit (BMK-2202, Vector Laboratories). All slides were storedovernight at 4 °C. Peroxidase and protein blocking was per-formed again as described above. The slides were incubated witha cytokeratin primary antibody (Z0622, rabbit, Dako, 1/50), thenstained with a polymer labeled secondary antibody (Alexa Flour488 goat anti-rabbit, A11034, Invitrogen) and DAPI. Immuno-fluorescent images were taken using a PM2000 microscope(HistoRX; magnification, �60). Representative images wereanalyzed for intensity of E-cadherin, vimentin, and Twist ex-pression using the Image J program (NIH, Bethesda, MD), andthe pixel values were averaged and graphically represented usingMicrosoft Excel:Mac v.x (Microsoft Corporation).

Migration Assays. Briefly, 2.5 � 104 cells were seeded on an8-�m-pore size Transwell filter insert (Corning Inc.) coated withECM (1:7.5) (Sigma). After 6 h of incubation at 37 °C and 5%CO2, cells adherent to the upper surface of the filter wereremoved using a cotton applicator. Cells were stained with 0.4%crystal violet dissolved in methanol, and the numbers of cells onthe bottom were counted. Data represent at least three exper-iments done in triplicate (mean � standard error).

1. Lin T, et al. (2005) p53 induces differentiation of mouse embryonic stem cells bysuppressing Nanog expression. Nat Cell Biol 7:165–171.

2. Chen S, et al. (2006) Self-renewal of embryonic stem cells by a small molecule. Proc NatlAcad Sci USA 103:17266–17271.

3. Swamynathan SK, et al. (2007) Conditional deletion of the mouse Klf4 gene results incorneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells. MolCell Biol 27:182–194.

4. Katiyar S, Jiao X, Wagner E, Lisanti MP, Pestell RG (2007) Somatic excision demonstratesc-Jun induces cellular migration and invasion through induction of stem cell factor. MolCell Biol 27:1356–1369.

5. Cheng GZ, et al. (2007) Twist transcriptionally up-regulates AKT2 in breast cancer cellsleading to increased migration, invasion, and resistance to paclitaxel. Cancer Res67:1979–1987.

6. Kwok WK, Ling MT, Yuen HF, Wong YC, Wang X (2007) Role of p14ARF in TWIST-mediated senescence in prostate epithelial cells. Carcinogenesis 28:2467–2475.

7. Aliouat-Denis CM, et al. (2005) p53-independent regulation of p21Waf1/Cip1 expres-sion and senescence by Chk2. Mol Cancer Res 3:627–634.

8. Wu K, et al. (2006) DACH1 is a cell fate determination factor that inhibits Cyclin D1 andbreast tumor growth. Mol Cell Biol 26:7116–7129.

9. Ju X, et al. (2007) Akt1 governs breast cancer progression in vivo. Proc Natl Acad Sci USA104:7438–7443.

10. Hulit J, et al. (2006) p27Kip1 repression of ErbB2-induced mammary tumor growth intransgenic mice involves Skp2 and Wnt/beta-catenin signaling. Cancer Res 66:8529–8541.

Liu et al. www.pnas.org/cgi/content/short/0910009106 1 of 6

http://www.pnas.org/cgi/content/short/0910009106

BA

Differentially regulated genesRas vs WT = 539 identified transcriptsMyc vs WT = 761 identified transcripts

417344195

Myc vs WTRas vs WT

21620303

Differentially regulated genesRas/p21+/+ vs Ras/p21-/- = 323 identified transcriptsMyc/p21+/+ vs Myc/p21-/- = 236 identified transcripts

Myc/p21+/+ vs Myc/p21-/-Ras/p21+/+ vs Ras/p21-/-

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JUPARHGEF1PKP1TIMP3EGR1ITPR1ATP1A1NRP1PLK2CTGFNUMBIRF6GRB7CDH10TCF2CLDN3ITGA4THBS1BCL6FRZBZO1CDH9CDH6HNF4ACDH1SLC27A2KRT17KRT18

ASNSCDH15CDKN1ACFHCTBP2CTNNB1CYP1B1GALK1HIF1AIL11LAMA5MMP14MMP2NOTCH1NOTCH3PDGFRAPMP22PROCRADAAMFRV-REL AVIMUPP1STAT3SRMSLPISIN3ARAF1PTGS1

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Fold Change

CAR2TACSTD1FLNAFOSBITPR1SERPINB5IRF6PCXF3VAMP8SAT1CDH1EGR1ATP1A1EGR2TGM2ITGB5PRKCZATF3KLF2CLDN3CDH10KRT18ITGA4ID2SLC27A2CDH6CTSHTGFB3DFFBRAB25FRZBZO1CDH15CDH4CDH9CDH1KRT8HNF4A

CDKN1ACOL6A1COL6A2CTBP1CTBP2CTSZDAB2EGR1ETS1HDAC2HIF1AHMGA2LAMA5LAMB1-1MTHFD2NOTCH1PLA2G7PPICRAF1ASNSCD68TNCTGF 1TCF4SRMSPARCSLC3A2SIN3A

Fig. S1. Microarray analysis of MMTV-Ha-Ras- and MMTV-c-Myc-induced tumors. (A) Venn diagram of genes differentially regulated between MMTV-Ras andMMTV-c-Myc compared to WT mammary gland. (B) Genes differentially regulated by MMTV-Ras and MMTV-c-Myc compared to WT mammary gland that overlapwith genes associated with EMT (13, 17). Genes upregulated in yellow and downregulated in blue relative to WT mammary gland (P � 0.05). Genes representedas gray were not differentially expressed at a significance of P � 0.05. (C) Venn diagram of genes differentially regulated by p21CIP1 in MMTV-Ras- andMMTV-c-Myc induced tumors. (D) Analysis of sample set enrichment scores (ASSESS) was used to classify the pathways differentially regulated by p21CIP1 inMMTV-Ras and (E) MMTV-c-Myc. Sample set enrichment scores concordant across each sample were used in a hierarchical cluster with complete linkage. Reddenotes positive enrichment and upregulation, blue denotes negative enrichment and down regulation. Pathways indicated in red illustrate upregulation ofEMT and LTHSC in p21CIP1�/� tumors.



D

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MMTV-HA-RasBROCKE_IL6KRETZSCHMAR_IL6_DIFFCANCER_NEOPLASTIC_META_UPADIP_DIFF_CLUSTER1HCC_SURVIVAL_GOOD_VS_POOR_DNUVB_NHEK3_C8GH_AUTOCRINE_UPMAMMARY_DEV_UPHSC_LTHSC_FETALHSC_LTHSC_SHAREDCANCER_UNDIFFERENTIATED_META_UPEMT_UPJECHLINGER_EMT_UPSANA_TNFA_ENDOTHELIAL_DNCELL_SURFACE_RECEPTOR_LINKED_SIGNAL_TRANSDUCTIONUVC_TTD_4HR_DNIL6_FIBRO_UPYANG_OSTECLASTS_SIGSTEMCELL_COMMON_DNHCC_SURVIVAL_GOOD_VS_POOR_UPNAB_LUNG_DNHOFMANN_MDS_CD34_LOW_RISKGN_CAMP_GRANULOSA_UP

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ALKPATHWAYIFN_BETA_GLIOMA_UPBYSTROM_IL5_DNDER_IFNG_UPADIP_HUMAN_UPNADLER_OBESITY_UPTGFBETA_ALL_UPSANA_TNFA_ENDOTHELIAL_DNHINATA_NFKB_UPIRITANI_ADPROX_VASCSHEPARD_POS_REG_OF_CELL_PROLIFERATIONYU_CMYC_DNHSC_LTHSC_ADULTRADAEVA_IFNA_UPG1_TO_S_CELL_CYCLE_REACTOMEBRUNO_IL3_DNKENNY_WNT_UPADIP_DIFF_CLUSTER2DNA_REPLICATION_REACTOMEIL1RPATHWAYUVC_HIGH_D3_DNUVC_TTD_8HR_DNUVC_XPCS_8HR_DNUVC_XPCS_ALL_DN

p21C

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Fig. S1. Continued.



B C DE-Cadherin

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p21CIP1+/+ p21CIP1-/- p21CIP1+/+ p21CIP1-/-

Red: E-Cadherin Green: CytokeratinBlue: DAPI

Red: Vimentin

Blue: DAPIGreen: Cytokeratin

Red: Twist

Blue: DAPIGreen: Cytokeratin

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Fig. S2. p21CIP1 suppresses EMT in MMTV-Ha-Ras and MMTV-c-Myc mice. (A) Representive immunohistochemical staining for E-cadherin (top panel), vimentin(middle panel), and Twist (bottom panel) in mammary tumors of MMTV-Ras p21CIP1�/�, MMTV-Ras/p21CIP1�/�, MMTV-c-Myc/p21CIP1�/� and MMTV-c-Myc/p21CIP1�/� mice. (B–D) Relative intensity of E-cadherin (B), vimentin (C), and Twist (D) protein abundance in murine mammary tissue showed by pixel value.



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Fig. S3. Twist induces epithelial to mesenchymal transition in human MCF10A mammary epithelial cells. (A) Twist induces colony formation of MCF10A in softagar. (B) Twist reduces S-phase in MCF10A cells determined by FACS assay. (C) Twist enhances migratory capability of MCF10A cell in a Transwell assay. (D) Twistinduces mesenchymal morphologies in 2D culture (panels 1 and 2) and induces collagen matrix invasion in 3D culture (panels 3 and 4). (E) Expression of theepithelial markers E-cadherin, �-catenin, and mesenchymal markers vimentin and N-cadherin were examined by immunoblotting in MCF10A-Twist and controlcells. (F) p21CIP1 antagonizes Twist-mediated repression of E-cadherin promoter activity. (G and H) p21CIP1 expression level (G) and promoter activity (H) wasinhibited by Twist.



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Fig. S4. p21CIP1 inhibits Ha-Ras-mediated induction of Twist. (A) Immunofluorescence staining of epithelial marker (ZO-1) and mesenchymal marker(N-Cadherin) in MCF10A-Ras and MCF10A-c-Myc. (B and C) p21CIP1 inhibits Twist promoter activity induced by Ras and c-Myc. All luciferase reporter assays werenormalized to Renilla Luciferase and represent � SEM (n � 3).


supporting information - pnas...2009/10/26 · microsoft excel:mac v.x (microsoft corporation)....

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