SI Text Western Blot Analysis, Immunofluorescence, and Immunohistochem- Quantitative Real-Time PCR

Supporting Information

Liu et al. 10.1073/pnas.0910009106 SI Text Western Blot Analysis, Immunofluorescence, and Immunohistochem- Quantitative Real-Time PCR. Oligonucleotides used for RT-PCR istry (IHC). IHC was following conducted as described in ref. 10. include: Human Twist: Forward: 5Ј- ggagtccgcagtcttacgag, Re- Briefly, after slides were rehydrated in graded alcohol, retrieved verse: 5Ј- cctctaccaggtcctccaga; Nanog: Forward: CA- for antigen using Citra (Biogenex) in a steamer, and blocked with GAAAAACCAGTGGTTGAAGACTAG, Reverse: GCAAT- peroxidase and goat serum, the slides were incubated with an GGATGCTGGGATACTC (1); Oct4: Forward: CTGTA- antibody specific for E-cadherin (#610182, BD Biosciences), GGGAGGGCTTCGGGCACTT, Reverse: CTGAGGGC- vimentin (M7020, Dako), or Twist (T6451, Sigma); all dilutions CAGGCAGGAGCACGAG (2); Sox2: Forward: GGCAGCTA- were 1:50. For the rabbit primary antibodies, slides were incu- CAGCATGATGCAGGAGC, Reverse: CTGGTCATGGAGT- bated with Envision System labeled polymer-HRP anti-rabbit TGTACTGCAGG (2); KLF4: Forward: TGCCAGA- secondary antibody (K4003, Dako) with TSA-plus Cyanine 5 CCAGATGCAGTCAC, Reverse: GTAGTGCCTGGTCAGT- system (NEL745001KT, Perkin-Elmer LAS). For the mouse TCATC (3); c-Myc: Forward: TGAGCCCCTAGTGCTGCAT, primary antibodies, slides were stained using the M.O.M. basic Reverse: AGCCCGACTCCGACCTCTT; 18s rRNA oligos (4). kit (BMK-2202, Vector Laboratories). All slides were stored overnight at 4 °C. Peroxidase and protein blocking was per- Cell Culture, Retroviral Infection, Luciferase Reporter Assay, siRNA formed again as described above. The slides were incubated with Transfection, and 3-D Culture. The Twist-specific siRNA target sequences is GGACAAGCTGAGCAAGATTCA (5, 6). The a cytokeratin primary antibody (Z0622, rabbit, Dako, 1/50), then p21CIP1 siRNA sequence is GCCTTAGTCTCAGTTTGGT- stained with a polymer labeled secondary antibody (Alexa Flour GTCTT (7). Twist and p21CIP1-specific siRNAs or control siR- 488 goat anti-rabbit, A11034, Invitrogen) and DAPI. Immuno- NAs (100 nM) were transfected by the Nucleofector technology fluorescent images were taken using a PM2000 microscope ϫ using Nucleofector Kit V and program T-024 (Amaxa Biosystems). (HistoRX; magnification, 60). Representative images were Transfection efficiency was monitored by non-silencing fluoresce- analyzed for intensity of E-cadherin, vimentin, and Twist ex- in-labeled siRNA from QIAGEN. For 3-D culture, 2,000 cells pression using the Image J program (NIH, Bethesda, MD), and mixed with growth factor reduced Matrigel matrix (volume ratio the pixel values were averaged and graphically represented using 1:1) were seeded into a four-well chamber and photographed Microsoft Excel:Mac v.x (Microsoft Corporation). according to a previously described protocol (8, 9). Migration Assays. Briefly, 2.5 ϫ 104 cells were seeded on an Mammosphere Formation and FACS Analysis of Stem Cell Surface 8-␮m-pore size Transwell filter insert (Corning Inc.) coated with Markers. Before labeling, the cells were blocked with normal ECM (1:7.5) (Sigma). After6hofincubation at 37 °C and 5% mouse IgG in 1/100 dilution for 30 min and then incubated with CO2, cells adherent to the upper surface of the filter were PE labeled mouse anti-human CD24 (1/5) (clone ML5, BD removed using a cotton applicator. Cells were stained with 0.4% PharMingen) and/or PE/Cy5 labeled rat anti human/mouse crystal violet dissolved in methanol, and the numbers of cells on CD44 (1/200) (clone IM7, BioLegend) for 1 h. All experiments the bottom were counted. Data represent at least three exper- were conducted at 4 °C. Cell sorting was performed on a iments done in triplicate (mean Ϯ standard error). FACSCalibur cell sorter (BD Biosciences). The data were analyzed with FlowJo software (Tree Star, Inc.).

1. Lin T, et al. (2005) p53 induces differentiation of mouse embryonic stem cells by 6. Kwok WK, Ling MT, Yuen HF, Wong YC, Wang X (2007) Role of p14ARF in TWIST- suppressing Nanog expression. Nat Cell Biol 7:165–171. mediated senescence in prostate epithelial cells. Carcinogenesis 28:2467–2475. 2. Chen S, et al. (2006) Self-renewal of embryonic stem cells by a small molecule. Proc Natl 7. Aliouat-Denis CM, et al. (2005) p53-independent regulation of p21Waf1/Cip1 expres- Acad Sci USA 103:17266–17271. sion and senescence by Chk2. Mol Cancer Res 3:627–634. 3. Swamynathan SK, et al. (2007) Conditional deletion of the mouse Klf4 gene results in 8. Wu K, et al. (2006) DACH1 is a cell fate determination factor that inhibits Cyclin D1 and corneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells. Mol breast tumor growth. Mol Cell Biol 26:7116–7129. Cell Biol 27:182–194. 9. Ju X, et al. (2007) Akt1 governs breast cancer progression in vivo. Proc Natl Acad Sci USA 4. Katiyar S, Jiao X, Wagner E, Lisanti MP, Pestell RG (2007) Somatic excision demonstrates 104:7438–7443. c-Jun induces cellular migration and invasion through induction of stem cell factor. Mol 10. Hulit J, et al. (2006) p27Kip1 repression of ErbB2-induced mammary tumor growth in Cell Biol 27:1356–1369. transgenic mice involves Skp2 and Wnt/beta-catenin signaling. Cancer Res 66:8529– 5. Cheng GZ, et al. (2007) Twist transcriptionally up-regulates AKT2 in breast cancer cells 8541. leading to increased migration, invasion, and resistance to paclitaxel. Cancer Res 67:1979–1987.

Liu et al. www.pnas.org/cgi/content/short/0910009106 1of6 A B Ras vs WT Myc vs WT

Ras Myc

ASNS CDKN1A CDH15 COL6A1 CDKN1A COL6A2 CFH CTBP1 CTBP2 CTBP2 195 344 417 CTNNB1 CTSZ CYP1B1 DAB2 GALK1 EGR1 HIF1A ETS1 IL11 HDAC2 LAMA5 HIF1A MMP14 HMGA2 MMP2 LAMA5 NOTCH1 LAMB1-1 NOTCH3 MTHFD2 PDGFRA NOTCH1

EMT UP PMP22 PLA2G7 Differentially regulated genes PROCR PPIC ADA RAF1 Ras vs WT = 539 identified transcripts AMFR ASNS V-REL A CD68 Myc vs WT = 761 identified transcripts VIM TNC UPP1 TGF 1 STAT3 TCF4 SRM SRM SLPI SPARC SIN3A SLC3A2 RAF1 SIN3A PTGS1

JUP CAR2 ARHGEF1 TACSTD1 PKP1 FLNA TIMP3 FOSB EGR1 ITPR1 ITPR1 SERPINB5 C ATP1A1 IRF6 NRP1 PCX F3 +/+ -/- +/+ -/- PLK2 Ras/p21 vs Ras/p21 Myc/p21 vs Myc/p21 CTGF VAMP8 NUMB SAT1 IRF6 CDH1 GRB7 EGR1 CDH10 ATP1A1 TCF2 EGR2 CLDN3 TGM2 ITGA4 ITGB5 THBS1 PRKCZ BCL6 ATF3 FRZB KLF2 ZO1 CLDN3 CDH9 CDH10

303 20 216 EMT DOWN CDH6 KRT18 HNF4A ITGA4 CDH1 ID2 SLC27A2 SLC27A2 KRT17 CDH6 KRT18 CTSH TGFB3 DFFB RAB25 FRZB ZO1 -70 +6 CDH15 Differentially regulated genes CDH4 +/+ -/- CDH9 Ras/p21 vs Ras/p21 = 323 identified transcripts CDH1 +/+ -/- KRT8 Myc/p21 vs Myc/p21 = 236 identified transcripts Fold Change HNF4A

Fig. S1. Microarray analysis of MMTV-Ha-Ras- and MMTV-c-Myc-induced tumors. (A) Venn diagram of genes differentially regulated between MMTV-Ras and MMTV-c-Myc compared to WT mammary gland. (B) Genes differentially regulated by MMTV-Ras and MMTV-c-Myc compared to WT mammary gland that overlap with genes associated with EMT (13, 17). Genes upregulated in yellow and downregulated in blue relative to WT mammary gland (P Ͻ 0.05). Genes represented as gray were not differentially expressed at a signiﬁcance of P Ͻ 0.05. (C) Venn diagram of genes differentially regulated by p21CIP1 in MMTV-Ras- and MMTV-c-Myc induced tumors. (D) Analysis of sample set enrichment scores (ASSESS) was used to classify the pathways differentially regulated by p21CIP1 in MMTV-Ras and (E) MMTV-c-Myc. Sample set enrichment scores concordant across each sample were used in a hierarchical cluster with complete linkage. Red denotes positive enrichment and upregulation, blue denotes negative enrichment and down regulation. Pathways indicated in red illustrate upregulation of EMT and LTHSC in p21CIP1Ϫ/Ϫ tumors.

Liu et al. www.pnas.org/cgi/content/short/0910009106 2of6 D Cip+/+ Cip-/- MMTV-HA-Ras p21 p21 BROCKE_IL6 KRETZSCHMAR_IL6_DIFF CANCER_NEOPLASTIC_META_UP ADIP_DIFF_CLUSTER1 HCC_SURVIVAL_GOOD_VS_POOR_DN UVB_NHEK3_C8 GH_AUTOCRINE_UP MAMMARY_DEV_UP HSC_LTHSC_FETAL HSC_LTHSC_SHARED CANCER_UNDIFFERENTIATED_META_UP EMT_UP JECHLINGER_EMT_UP SANA_TNFA_ENDOTHELIAL_DN CELL_SURFACE_RECEPTOR_LINKED_SIGNAL_TRANSDUCTION UVC_TTD_4HR_DN IL6_FIBRO_UP -1.09 YANG_OSTECLASTS_SIG STEMCELL_COMMON_DN HCC_SURVIVAL_GOOD_VS_POOR_UP 0 NAB_LUNG_DN HOFMANN_MDS_CD34_LOW_RISK GN_CAMP_GRANULOSA_UP +1.09

E Cip+/+ Cip-/- MMTV-c-Myc p21 p21 ALKPATHWAY IFN_BETA_GLIOMA_UP BYSTROM_IL5_DN DER_IFNG_UP ADIP_HUMAN_UP NADLER_OBESITY_UP TGFBETA_ALL_UP SANA_TNFA_ENDOTHELIAL_DN HINATA_NFKB_UP IRITANI_ADPROX_VASC SHEPARD_POS_REG_OF_CELL_PROLIFE RATION YU_CMYC_DN HSC_LTHSC_ADULT RADAEVA_IFNA_UP G1_TO_S_CELL_CYCLE_REACTOME BRUNO_IL3_DN KENNY_WNT_UP ADIP_DIFF_CLUSTER2 DNA_REPLICATION_REACTOME IL1RPATHWAY UVC_HIGH_D3_DN UVC_TTD_8HR_DN UVC_XPCS_8HR_DN UVC_XPCS_ALL_DN

Fig. S1. Continued.

Liu et al. www.pnas.org/cgi/content/short/0910009106 3of6 A MMTV-Ras MMTV-Myc

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

Red: E-Cadherin Green: Cytokeratin Blue: DAPI E-Cadherin

Red: Vimentin Green: Cytokeratin Blue: DAPI Vimentin

Red: Twist Green: Cytokeratin

Twist Blue: DAPI

50 M 50 M

BCD E-Cadherin Vimentin Twist 80 160 100 140 80 60 120 100 60 40 80 60 40 20 40 20 Imagel J pixel value pixel J Imagel Imagel J pixel value Imagel Imagel J pixel value 20 0 0 0 Cip1-/- Cip1-/- Cip1-/- Cip1-/- Cip1-/- Cip1-/- Cip1+/+ Cip1+/+ Cip1+/+ Cip1+/+ Cip1+/+ Cip1+/+ p21 p21 p21 p21 p21 p21 p21 p21 p21 p21 p21 p21 Ras Myc Ras Myc Ras Myc

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 p21CIP1ϩ/ϩ, MMTV-Ras/p21CIP1Ϫ/Ϫ, MMTV-c-Myc/p21CIP1ϩ/ϩ and MMTV-c-Myc/ p21CIP1Ϫ/Ϫ mice. (B–D) Relative intensity of E-cadherin (B), vimentin (C), and Twist (D) protein abundance in murine mammary tissue showed by pixel value.

Liu et al. www.pnas.org/cgi/content/short/0910009106 4of6 ABC 100 100 200 G1 Serum-free S Serum 80 12.5 80 25.6 G2/M 160

Vector 60 60 120

40 40 80 distribution Colonies/dish Colonies/dish %

Twist 20 20 40 Migrated cells/view Migrated 0 0 0

Vector Twist Vector Twist Vector Twist DE 2-D 3-D Vector Twist 1 3 -myc-Twist

E-Cadherin Vector -Catenin Epithelial

-Vimentin MCF10A 2 4

-N-Cadherin

Twist -actin Mesenchymal 50 M 50 M

FH E-Cad(-108) Luc G -108 p21-Luc E-Cad(-108)-EboxMut Luc -108 -2400 1.4 1.2 1.2 1.0 MCF10A 1.0 0.8 Vector Twist 0.8 * 0.6 0.6 -myc-Twist induction 0.4 0.4 CIP1 Fold induction -p21 Fold 0.2 0.2 -actin 0 0 Twist - + + - + + Twist - p21CIP1 --+ --+ WT Luc Mut Luc

Fig. S3. Twist induces epithelial to mesenchymal transition in human MCF10A mammary epithelial cells. (A) Twist induces colony formation of MCF10A in soft agar. (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) Twist induces mesenchymal morphologies in 2D culture (panels 1 and 2) and induces collagen matrix invasion in 3D culture (panels 3 and 4). (E) Expression of the epithelial markers E-cadherin, ␤-catenin, and mesenchymal markers vimentin and N-cadherin were examined by immunoblotting in MCF10A-Twist and control cells. (F) p21CIP1 antagonizes Twist-mediated repression of E-cadherin promoter activity. (G and H) p21CIP1 expression level (G) and promoter activity (H) was inhibited by Twist.

Liu et al. www.pnas.org/cgi/content/short/0910009106 5of6 A MCF10A MCF10A

Vector Ras Myc Vector Ras Myc ZO-1 N-Cadherin

50 M 50 M

B Twist-LUC C Twist-LUC -1,900 -1,900 LUC LUC 4 4 HEK293 HEK293

3 3

2 2 Fold induction 1 Fold induction 1

0 0 Vec - - Vec - - Myc - - ++ Ras - - ++ p21CIP1 - - p21CIP1 - -

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 were normalized to Renilla Luciferase and represent Ϯ SEM (n Ն 3).

Liu et al. www.pnas.org/cgi/content/short/0910009106 6of6