Cell Reports, Volume 17

Supplemental Information

WIP Drives Tumor Progression through

YAP/TAZ-Dependent Autonomous Cell Growth

Ricardo Gargini, Maribel Escoll, Esther García, Ramón García-Escudero, FranciscoWandosell, and Inés María Antón

SUPPLEMENTAL FIGURES AND SUPPLEMENTAL LEGEND TO THE FIGURES

Figure S1. Related to Figure 1. WIP elimination impairs tumor growth. (A) Western blot analysis of WIP and GAPDH (loading control) in a panel of tumor cell lines. (B) WIP mRNA is overexpressed in the GB mesenchymal compared to classical, neural and proneural subtype. Significant differences were calculated using Student’s t-test, and the p value indicated. (C-F) Growth curves measured by MTT assay at the indicated times of the cell lines MDA-MB-231 (C), Hs578T (D), U87-MG (E) and U373-MG (F) expressing two shRNA specific for WIP (shWIP88 and shWIP66) and a control shRNA (n = 4). (G, H) CD44Low/CD24High and CD44High/CD24Low populations of MDA-MB-231 knocked down for WIP or controls were purified by FACS. (G) Western blot analysis of the expression of WIP and GAPDH control. (H) Limiting dilution analysis. Sphere formation ability was quantified at 6 days and tumor sphere formation frequency was determined by ELDA (n = 6). (C-F) Data shown as mean ± SEM.

Figure S2. Related to Figure 2. WIP expression is associated with the amount of YAP and TAZ and is essential for the maintenance of stem/mesenchymal phenotype in solid tumors. (A) Western blot analysis of YAP/TAZ, WIP and GAPDH (loading control) in a panel of cell lines of different tumor types. (B) Quantification of the correlation between the expression of YAP and TAZ vs WIP. (C) Western blot analysis of E-cadherin, YAP/TAZ, CTGF, survivin, WIP and actin (loading control) of two lines with stem/mesenchymal phenotype (MDA-MB-231 and Hs578T; basal type B) and two lines with epithelial phenotype (T47D and MCF-7; luminal type). (D) Flow cytometry analysis of MDA-MB-231 and Hs578T knockdown for WIP or control representing the percentage of cells that show stem-like characteristics (CD44high/CD24low). (E) Representative confocal images of YAP/TAZ (green), DAPI (blue) and phalloidin (red) in MDA-MB-231 cells after interference with shcontrol or shWIP in

stem conditions; bars, 50 µm. (F-H) Box plots showing relative WIP (F), TAZ (G) and YAP1 (H) mRNA levels in the different tumor types, extracted from CCLE_Expression_Entrez_2012-10-18.res, with gene-centric robust multiarray analysis-normalized mRNA expression data. The number of cell lines for each tumor type analyzed is indicated in parentheses. (I) Western blot analysis of YAP/TAZ, WIP and GAPDH (loading control) in MDA-MB-231 and two hematological cancer cell lines (Jurkat and U-937) knocked down for WIP or controls. Growth curves of Jurkat (J) and U-937 (K) cell lines measured by trypan blue. NS, not significant. (D, J, K) Data shown as mean ± SEM; n = 3.

Figure S3. Related to Figure 3. Increased WIP expression stimulates growth and induces stemness in a YAP/TAZ-dependent manner. (A, B) Astrocytes overexpressing GFP or WIP-GFP were transduced with lentivirus encoding shRNA specific against TAZ, YAP or control. (A) Western blot analysis of YAP/TAZ and GAPDH. (B) Representative images of the astrocytes analyzed in the experiment; bars, 10 µm. (C, D) Growth curve measured by MTT assay of human primary astrocytes (C) and a MCF10A breast cell line (D), both overexpressing GFP or WIP full length and mutants of the NCK binding domain (LV-WIP-ΔNBD) or the actin binding domain (LV-WIP-Δ42-53). (C, D) Data shown as mean ± SEM; n = 4.

Figure S4. Related to Figure 4. WIP coordinates survival and proliferation to drive the invasiveness in a YAP/TAZ-dependent manner. (A-C) MDA-MB-468 cells were transduced with lentivirus encoding GFP, WIP-GFP, WIP-ΔNBD or WIP-Δ42-53. (A) Confocal images of staining for GFP (cyan), Ki67 (red), YAP/TAZ (green) and nuclei (DAPI, blue) of invasive/non-invasive structures in 3D-Matrigel; bars, 25 µm. (B) Percentage of cells that showed positive staining for Ki67 or (C) nuclear localization of YAP/TAZ. (D, E) MDA-MB-468 cells transduced with lentivirus encoding GFP or GFP-WIP and then with shcontrol or shWIP were cultured in 3D-Matrigel and stained with ethidium bromide. (D) Representative phase contrast images of the structures; bars, 25 µm. (E) Percentage of structures showing ethidium bromide-positive staining. (B, C, E) Data shown as mean ± SEM; n = 3.

Figure S5. Related to Figure 5. WIP stabilizes YAP/TAZ and induces resistance to Lats- and Mst-mediated degradation. (A) TAZ mRNA levels measured by qRT-PCR normalized to GAPDH expression in GB4, GB5 and GB8 cells knocked down for WIP or controls (NS, not significant). (B) Analysis of the percentage of apoptosis induction measured by annexinV/7AAD, as determined by flow cytometry in MDA-MB-231 cells infected with lentivirus shcontrol or shWIP66 and transfected with pcDNA, TAZ-wt or TAZ-S311A (a degradation-insensitive mutant). (C, D) Flow cytometry quantification of TEAD-dependent transcription by cherry protein under the TEAD sequence promoter (C) or β-catenin-dependent transcription by TOP/FOP-GFP (D) in MDA-MB-231 cells infected with lentivirus shcontrol or shWIP66, and then transfected with pcDNA or TAZ-S311A. (E, F) Astrocytes expressing GFP or WIP-GFP were transduced with lentivirus encoding Lats1, Mst2, or both. (E) Representative images of the growth as spheres (bars, 50 µm) and quantification of sphere number. (F) Western blot analysis of pLats1, Lats1, pYAP, YAP/TAZ, Mst2, survivin, WIP-GFP and GAPDH (loading control). (G, H) MDA-MB-231 cells knocked down for WIP or control, and transduced with shcontrol or shLats1/2-encoding lentivirus and cultured in stem conditions. (G) Western blot analysis of Lats1, Lats2, YAP/TAZ, WIP and GAPDH. (H) Quantification of sphere and cell number. (I, J) Astrocytes overexpressing YAP-wt or YAP-S5A were transduced with lentivirus encoding shRNAs to WIP, TAZ or control and grown in stem conditions. (I) Western blot analysis of YAP/TAZ and GAPDH (loading control). (J) Representative images and quantification of sphere numbers. (A-E, H, J) Data shown as mean ± SEM; n = 3.

Figure S6. Related to Figure 6. WIP regulates YAP/TAZ stability by controlling the endocytic/endosomal system. (A, B) GB4, GB5 and GB8 cells were culured with vehicle (control), monensin (10 µM), EIPA (50 µM), nigericin (5 µM) or salinomycin (10 µM) for 24 h. (A) Western blot analysis of β-catenin, YAP, TAZ, WIP and GAPDH (loading control) and quantification of spheres (B) after 6 days in culture in stem conditions. (C-E) Flow cytometry analysis of MDA-MB-231 and GB4 cells transduced with lentivirus encoding shWIP66 and shcontrol, cultured in stem conditions and stained with acridine orange (C), Magic Red (D) or LysoSensor (E). (F, G) MDA-MB-231 cells transduced with lentivirus encoding shRNAs to WIP, β-catenin, TAZ or control were grown in stem conditions. (F) Western blot analysis of β-catenin, TAZ and WIP and GAPDH (loading control) and quantification of sphere number (G), after 6 days culture in stem conditions. (H) Western blot analysis of HEK293 cells with WIP interference or

control, transfected with empty plasmid (pCIneo) or mutants that prevent correct APC/axin/β-GSK3 destruction complex formation, β-catenin S33Y (active β-catenin) or APC truncated. (I, J) Three colon carcinoma cell lines bearing mutations in APC (HT29, SW480 and SW620), transduced with lentivirus encoding shWIP66 or shcontrol, were cultured in stem conditions for 6 days. (I) Western blot analysis of TAZ, WIP and GAPDH (loading control) and quantification of cell number (J); NS, not significant. (K-L) Western blot analysis of YAP/TAZ, WIP and GAPDH (loading control) of MDA-MB-231 cells knocked down for WIP or controls after 6 days culture in stem conditions, followed by 24 h alone or with indicated concentrations of bafilomycin (K) or chloroquine (L). (B-E, G, J) Data shown as mean ± SEM; n = 3.

Figure S7. Related to Figure 7. WIP controls Rac/PAK activities that regulate the degradation complex by sequestration and mediate YAP/TAZ stabilization. (A) Western blot analysis of astrocytes expressing GFP or WIP-GFP, cultured in stem conditions for 3 days and treated for 24 h with vehicle (DMSO, control), NSC23766 (Rac inhibitor, 50 µM), casin (Cdc42 inhibitor, 5 µM) or Y16 (Rho inhibitor, 30 µM). (B-C) Glioblastoma cells GB4, GB5 and GB8 were cultured in stem conditions for 6 days and treated for 24 h with vehicle (control), NSC23766, casin or Y16 as in (A). (B) Western blot analysis of TAZ, WIP and GAPDH (loading control) and quantification of cell number (C). (D) Western blot analysis of YAP/TAZ, Rac1, WIP and GAPDH (loading control) in MCF10A cells transformed with RAC1-V12, then transduced with lentivirus encoding shRNA to WIP, TAZ or control, and cultured 6 days in stem conditions. (E) Western blot analysis of pSer144-PAK1, TAZ, WIP and GAPDH (loading control) in GB4, GB5 and GB8 glioblastoma explant cells transduced with lentivirus encoding shRNAs against WIP or control, after 6 days culture in stem conditions. (F-H) GB4, GB5 and GB8 cells were cultured in stem conditions (6 days) and treated for 24 h with vehicle (DMSO, control), IPA-3 (10 or 20 µM) (H) or SMIFH2 (15 or 30 µM) (I). (F, G) Western blot analysis of TAZ and GAPDH (loading control) and quantification of sphere number (H). (I-J) FACS separation of CD44Low/CD24High cells from CD44High/CD24Low cells after incubation with inhibitors of PAK (IPA-3, 10 µM), formins (SMIFH2, 30 µM) or actin polymerization (LatA1, latrunculin A1, 1 µM). (I) Western blot analysis of ABCβ-catenin, YAP/TAZ, survivin and GAPDH levels (loading control) and quantification of sphere number (J). (C, H, J) Data shown as mean ± SEM; n = 3.

SUPPLEMENTAL EXPERIMENTAL PROCEDURES Reagents and plasmids

The reagents acridine orange, propidium iodide, chloroquine, monensin, salinomicyn, nigericin, puromycin, hexadimethrine bromide (polybrene), saponin and 3-[4,5-dimethylthiazo-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma. We also used MG132 (10 µM), latrunculin A (all from Calbiochem), and triciribine (AKT-i, 10 µM), GDC0941 (PI3K-i, 1 µM), AKT inhibitor V (AKT-i, 5 µM), U0126 (MEK-i, 10 µM), PD98059 (MEKi, 20 µM), H89 (PKA-i, 10 µM), PP2 (Src family tyrosine kinases, 2 µM), imatinib (1 µM), dasatinib (0.1 µM), Y27632 (ROCK-i, 10 µM), NSC23766 (Rac-i, 50 µM), casin (Cdc42-i, 5 µM), Y16 (Rho-i, 30 µM), IPA-3 (PAK-i, 10 µM), GW4869 (sphingomyelinases-i, 2 µM), wiskostatin and 187-1 (N-WAPS-i, 10 and 1 µM, respectively), GM6001 (MMPs-i, 25 µM), CK666 and CK869 (ARP2/3-i 50 µM), SMIFH2 (mDIA-i 30 µM), cytochalasin (1 µM), bafilomycin, EIPA and jasplakinolide were purchased from Tocris. EGF (20 ng/ml) and WNT3a (80 ng/ml) were from Peprotech. Plasmids for overexpression of Rab5QL (#29688), Hrs (#29685), APC truncated (#16510) and β-catenin-S33Y (#19286) were obtained from Addgene. Cell culture

The cell lines MCF-7, T47D, MDA-MB-468, MDA-MB-231, Hs578T, HN19, Detroit, Fadu, HacaT, HT29, HCT116, SW480, SW620, Hs-683, U87-MG and U373-MG were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 0.1% penicillin (100 U/ml) and streptomycin (100 µg/ml). Jurkat and U937 cells lines were maintained in RPMI supplemented as above. Primary human astrocytes were cultured following supplier’s instructions (ScienCell Research Laboratories). MCF-10A cells were cultured in DMEM:F12 media (1:1) supplemented with 10 µg/ml insulin (Gibco/Invitrogen), 20 ng/ml EGF (Peprotech), 0.5 µg/ml hydrocortisone (Calbiochem), cholera toxin 100 ng/ml (Sigma) and 5% horse serum (Gibco/Invitrogen) at 37ºC with 7% CO2 and 97% relative humidity (Debnath et al. 2003).

For stem culture conditions, we used DMEM:F12 with FGF-2 (20 ng/ml), EGF-2 (20 ng/ml), heparin, B27 (without vitamin A for breast cells) and N2 supplements (for glial cells), 0.1% penicillin (100 U/ml) and streptomycin (100 µg/ml). Tumor sphere cultures were as described (Gargini et al. 2015, Ponti et al. 2005) using the medium composition described above. Tumor spheres were cultured for 4-7 days and those >50 µm diameter were counted. For serial passage, tumor spheres were harvested using 70 µm cell strainers after 6 days culture. The tumor spheres were trypsin-dissociated to single cells, and 500/1000 dissociated cells were plated in a 24-well plate and cultured for 4-7 days.

Limiting dilution assay was essentially as described (Hu & Smyth 2009). After shcontrol and shWIP66 lentivirus infection, cells were separated by FACS, and the CD44High/CD24Low and CD24High/CD44Low populations were seeded at different dilutions, cultured (6 days) and quantified. Data and statistics were calculated using Extreme Limiting Dilution Analysis (ELDA) software (http://bioinf.wehi.edu.au/software/limdil/index.html) (Hu & Smyth 2009). Antibodies and Western blot

Cells were lysed in 50 mM Tris (pH 7.5), 300 mM NaCl, 0.5% SDS, and 1% Triton X-100. From each sample, 30 µg of total protein (quantified with DC protein assay, Bio-Rad) was resolved on 10, 12 or 14% Tris-glycine polyacrylamide gels and transferred to nitrocellulose membranes. Blots were probed with antibodies to GFP, β-tubulin, actin and Flag2 (Sigma), E-cadherin, caveolin1, Rac1, TAZ, β-GSK3, EEA1, nestin and β-catenin (BD Transduction), ABC-β-catenin (Millipore), cleaved caspase 3 (casp3 Act), pSer144/149-PAK1, pThr202/Tyr204-ERK, pT1089-Lats1, Mst2, Lats2, Lats1, cyclin D1, axin1, p127YAP, YAP/TAZ and GAPDH (Cell Signaling Technology), α/β-GSK3 (Invitrogen), survivin, CTGF, WIP, and total ERK1/2 (Santa Cruz Biotechnology). Secondary antibodies used were horseradish peroxidase-conjugated anti-rabbit or -mouse IgG (Dako). Survival analyses

WIP gene expression and follow-up overall survival data from human glioblastoma tumors corresponding to TCGA and Freije data sets were downloaded respectively from cBioPortal (http://www.cbioportal.org/) and GEO databases (http://www.ncbi.nlm.nih.gov/gds); the Freije GEO identifier is GSE4412. Kaplan-Meier survival curves were done following patient stratification using WIP expression values. TCGA patients (n = 588) were stratified in three groups using WIP z-score values as thresholds. Freije patients (n = 59) were stratified in two equal groups using the median WIP expression value as the cut-off point. Significance differences in survival between groups were calculated using the log rank test.

Correlation between continuous WIP expression values and overall survival was analyzed using univariate Cox regression analysis. Cox gives a hazard ratio value that computes the ratio of incidence rates of patients with high versus low WIP expression.

3D acinar morphogenesis assay and 3D indirect immunofluorescence microscopy For 3D cultures, MDA-MB-231 or MDA-MB-468 cells were cultured in Matrigel, as described

(Xiang & Muthuswamy 2006), for 6 days before invasion was quantified. To visualize morphological changes in acinar structures, multiple DIC images were acquired using a Nikon TE2000E inverted fluorescence microscope equipped with a DIC analyzer. Between 200-300 acini were scored per condition in each experiment. 3D cultures were stained with ethidium bromide (Invitrogen) to visualize cell death-associated DNA fragmentation and of nuclear envelope (Muranen et al. 2012).

Immunofluorescence analysis of 3D cultures was as described (Debnath et al. 2002). Briefly, cells grown in 3D cultures were washed with PBS and fixed in 4% paraformaldehyde (PFA; Sigma) in PBS (20 min, room temperature; RT). Cells were then rinsed 3 times (10 min each) with PBS:glycine (100 mM) and permeabilized with 0.5% Triton X-100 in PBS (10 min), blocked with 10% goat serum (Sigma) in IF buffer (130 mM NaCl, 7 mM Na2HPO4, 3.5 mM NaH2PO4, 7.7 mM NaN3, 0.1% BSA, 0.2% Triton X-100, 0.05% Tween-20) (1 h, RT) and incubated with primary antibodies in the same solution (overnight, 4ºC). Cells were washed 3 times, (20 min each) with IF buffer. When required, cells were incubated with Alexa-conjugated secondary antibodies (1 h, RT) (Molecular Probes). Cells were washed twice with IF buffer, once with PBS, then incubated with DAPI (1:2000; Sigma; 20 min). Cells were mounted in Fluoromount-G mounting medium (Southern Biotech). Antibodies included anti-YAP/TAZ (1:100; Cell Signaling), -integrin α6 (1:200; BD Biosciences) and -Ki67 (1:200; MiB1 Dako). Phalloidin was used to stain F-actin (1:100; AlexaFluor TRITC; Invitrogen). Intracranial tumor assay

The protocol was approved by the Committee on the Ethics of Animal Experiments of the CBMSO institutional Biosafety and the Institutional Committee of the Comunidad Autonoma de Madrid (CAM; ref PROEX341-15, associated to project SAF2015-70368-R).

All surgery was performed under isofluorane gas anesthesia, and all efforts were made to minimize suffering. Cells (2 x 105) were inoculated stereotactically into the corpus striatum of the right brain hemisphere (1 mm anterior and 1.8 mm lateral to the bregma; 2.5 mm intraparenchymal) of 9-week-old NOD-SCID mice (Charles River Laboratories). Mice were euthanized when they presented neurological symptoms or significant weight loss. Tumors were obtained surgically and fixed with 4% PFA. 2D indirect immunofluorescence microscopy

Cells were attached to poly-L-lysine (0.0033%, Sigma)-coated glass coverslips, fixed with 4% PFA, then permeabilized with 0.1% Triton X-100 in PBS (10 min) and blocked with 0.1% Triton X-100, 1% FBS in PBS (20 min). Where indicated, cells were first permeabilized with RB buffer plus digitonin (65 µg/ml) or saponin (5 min, RT), fixed with 4% PFA (10 min), and incubated with PBS-0.2% Triton X-100 (5 min). Cells were incubated with the following antibodies (overnight, 4°C): anti-WIP (1:200, Santa Cruz Biotechnology), -β-GSK3 (1:100), -integrin α6 (1:200), -CD63 (1:200)(all three from BD Transduction) and -YAP/TAZ (1:200, Cell Signaling). Coverslips were washed three times and incubated with the appropriate secondary antibody (1 h, RT): donkey anti-rabbit/-mouse IgG labeled with Alexa488, Alexa555 or Alexa647 (1:500, Invitrogen), phalloidin-TRITC (1:200, Invitrogen), and mounted in Fluoromount G. For LysoTracker staining, live cells were incubated in pre-warmed culture medium with diluted LysoTracker Red DND-99 (1:1000, Invitrogen; 5 min, 37ºC), quickly washed twice with PBS, and fixed with 4% PFA (15 min, RT). Nuclei were DAPI-counterstained and viewed on an Axiovert200 microscope (Zeiss). Flow cytometry analysis and sorting

For FACS analysis, tumor spheres were digested with 0.025% trypsin (5 min, 37ºC) to dissociate them into single cells, then stained with anti-CD44 (G44-26), -CD24 (ML5 BD; both from Bioscience) and -CD133 (Miltenyi) antibodies or apoptosis markers annexinV-FITC or -Alexa647 and 7-AAD [7-amino-actinomycin D] (all from BD Biosciences) according to manufacturer’s guidelines, and were analyzed by flow cytometry on a FACSCalibur (BD Bioscience).

Endosomal function was analyzed by flow cytometry. MDA-MB-231 or GB4 cells were incubated with the endosomal pH-sensitive probe acridine orange (1 µM; Sigma; 30 min, 37ºC) (Degtyarev et al. 2008), LysoSensor Green DND-189 (to measure pH of acidic organelles qualitatively) (1 µM; Molecular Probes; 20 min, 37ºC) (Lu et al. 2013) and Magic Red (Immunochemistry Technologies). Magic Red reagent was diluted 1:365 in PBS, then added at a 1:10 dilution to cell culture medium for 10 min. Cells were resuspended in PBS and analyzed in a FACScalibur.

For flow cytometry-based transferrin uptake assays, cells were incubated with 10 µg/ml Alexa647-transferrin (30 min, 4°C), followed by incubation at 37°C for various time intervals in the continuous presence of Alexa647-transferrin. To measure plasma membrane-associated transferrin receptor, cells were washed and terminated experiment by pelleting and resuspension in 4% PFA. Cell-associated Alexa647-transferrin was determined by flow cytometry on a FACScalibur.

Lentiviral and retroviral vector production and infection Pseudotyped lentivectors were produced using reagents and protocols as reported (Gargini et al.

2015). The 293T cells were transiently cotransfected with 5 µg of appropriate lentivector plasmid, 5 µg packaging plasmid pCMVdR8.74 and 2 µg VSV-G envelope protein plasmid pMD2G (both from Addgene) using Lipofectamine Plus reagent (Invitrogen). Lentivector shRNA control, WIP, YAP, TAZ, β-catenin were from Sigma-Aldrich (MISSION shRNA). The lentiviruses that encode GFP, full-length WIP (WIP-GFP), WIP lacking the Nck binding site (WIP-GFPΔNCK) and WIP lacking the actin binding site (WIP-GFPΔ42-53) were produced in collaboration with Gareth E. Jones (King’s College, London, UK) (Banon-Rodriguez et al., 2013). TCF/LEF reporter driving expression in the GFP lentivector (TOP-GFP) was a gift from Laurie Ailles (Reya et al. 2003). The TEAD reporter assay was performed with the pLL3.7 K122-ires-GFP-TEAD-responsive-H2B mCherry reporter, a gift from Yutaka Hata (Addgene plasmid #68714). A lentiviral RNAi vector encoding miRNA to human LATS2 and LATS1 were from Addgene (#52085). The lentivirus encoding flag-mDia2 was the kind gift of Robert Grosse (Baarlink et al. 2013) and the lentivectors that express Lats1 and Mst2 were a generous gift from Xiaolong Yang (Queen’s University, Kingston, Ontario K7L 3N6, Canada) (Beausejour et al. 2003). Retroviral vectors used were pBabe-GFP and pMX-GFP-RAC-G12V (Addgene #14567). Retrovirus supernatant was prepared by transfection of phoenix-Ampho cells (Garry Nolan, Dept Microbiology and Immunology, Stanford University, Stanford, CA) with 5 µg of each plasmid using Lipofectamine plus. Cells were infected in the presence of polybrene (4 µg/ml) and selected with puromycin (0.5-1.5 µg/ml). MVB sequestration, digitonin and protease protection assay

Proteinase K protection assays were performed as described (Taelman et al. 2010, Vanlandingham & Ceresa 2009). MDA-MB-231 cells transduced with lentivirus shcontrol or shWIP66, or inhibitor-treated as indicated for each experiment, were trypsinized from a 10 cm culture plate and pelleted. Cells were resuspended in RB buffer (100 mM potassium phosphate (pH 6.7), 5 mM MgCl2, and 250 mM sucrose) containing 65 µg/ml digitonin and incubated (5 min, RT, followed by 30 min on ice). Digitonin was removed by centrifugation (16,100 xg; 5 min, RT), permeabilized cells were resuspended in RB buffer without digitonin and divided into Eppendorf tubes containing reagents to generate final concentrations of 1 µg/ml Proteinase K (Invitrogen), plus 0.1% Triton X-100 when indicated. After incubation (10 min, RT), the reaction was terminated by adding 20 mM protease PMSF preheated in 5X loading buffer, and heated (95ºC, 10 min) before electrophoresis. Soft agar assay

To evaluate the tumorigenic potential of cells after specific shRNA inhibition, viable cells (2 × 104/well) were plated in soft agar in 6-well plates. Briefly, the base layer was made by mixing equal volumes of sterile 1% agar cooled to 40°C, and 2× proliferation for a final solution of 0.5% agar in 1× stem culture medium. For the top layer, agar was diluted to 0.7% in distilled water, cooled to 40°C and mixed in equal proportions with 2× DMEM. Cells were added immediately to the mix to yield a final solution of 0.35% agar in 1× DMEM containing 104 cells/ml. Cells were cultured (10 days, 37°C, 5% CO2 in a humidified atmosphere) and viable colonies were stained with 1 ml/well 600 mg/ml MTT, photographed, and counted using ImageJ software (http://rsbweb.nih.gov/ij/). Quantitative real-time PCR assays Total RNA was prepared with the RNeasy kit (Qiagen). Complementary DNA was prepared using a TaqMan reverse-transcription kit, and real-time PCR with TaqMan PCR mixture (Applied Biosystems) according to standard protocols (Zhang et al. 2011). The Taqman primers for human TAZ (Hs00210007_m1, WWTR1) and human GAPDH (Hs99999905_m1) were purchased from Applied Biosystems. TAZ expression in cells was normalized to GAPDH gene expression.SUPPLEMENTAL

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