Supplemental Data YAP1 Increases Organ Size and Expands Undifferentiated Progenitor Cells

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1 Supplemental Data YAP1 Increases Organ Size and Expands Undifferentiated Progenitor Cells S1 Fernando D. Camargo, Sumita Gokhale, Jonathan B. Johnnidis, Dongdong Fu, George W. Bell, Rudolf Jaenisch, and Thijn R. Brummelkamp Supplemental Experimental Procedures Generation and Manipulation of YAP1-Inducible Mice Human YAP1 was PCR amplified, mutagenized to introduce a serine to alanine mutation at position 127, and cloned into the unique EcoRI restriction enzyme site of pbs31 [S1]. The pbs31-yap1-s127a plasmid was coelectroporated with a Flpe recombinase expression vector into V6.5 ES cells that were expressing the M2rtTA tetracycline-responsive transactivator under control of the ROSA26 promoter [S2]. Targeted ES cell clones were selected with hygromycin, expanded, and analyzed for correct integration at the collagen locus by Southern blot analysis with an external ColA1 3 0 probe. Two independently correctly targeted clones were injected into blastocysts to generate chimeric mice. Transgene expression was induced by feeding the mice 2 mg/ml doxycycline in their drinking water supplemented with 10 mg/ml sucrose. For liver-specific activation of YAP1, mice were crossed with animals expressing the tta tetracycline transactivator from the liver activator protein (LAP) promoter. Breeding of these mice was done in the presence of 2 mg/ml doxycycline in drinking water supplemented with 10 mg/ml sucrose until the time of weaning, where normal water was administered. For the experiments involving g-secretase inhibition, mice were daily injected intraperitoneally with 16 mmol/kg dipenzazepine (DBZ) suspended in 0.5% (w/v) Methocel E4M and Tween-80 in water. Dox was administered to these mice 1 day after the initiation of DBZ treatment and animals were analyzed 4 days later. Western Blot Analysis Western blots were performed with whole-cell extracts, separated on 4% 12% Novex Bis-Tris gels, and transferred to polyvinylidine difuoride membranes (Millipore). Intestinal epithelial cells were isolated by incubation of segments of the mouse intestine at 37 Cin a buffer containing 3 mm ethylenediamine tetraacetic acid (EDTA) and 0.1 mm dithiothreitol (DTT) in phosphate-buffered saline (PBS) for 30 min. Western blots were probed with the following antibodies: anti-yap1, Cell Signaling, rabbit polyclonal, (1:1000); anti-actin, Santa Cruz C-11, goat polyclonal (1:1000); anti-pcna, Santa Cruz PC-10, mouse monoclonal (1:1000); anti-bclxl, Cell Signaling 2762, rabbit polyclonal (1:1000) and anti-cyclind1, Santa Cruz H- 295, rabbit polyclonal (1:1000). Nuclear and cytoplasmic extracts were generated by resuspending cells in four packed volumes of cytoplasmic buffer (10 mm HEPES [ph 7.9]/10 mm NaCL/0.1 mm EDTA/5% glycerol/1.5 mm MgCl 2, 10 mm EDTA, and protease inhibitors). The lysate was centrifuged ( g, 10 min) and the supernatant was collected. The pellet was resuspended in 2.5 packed cell volumes of nuclear buffer (identical to cytoplasmic buffer but the concentration of NaCl was raised to 0.42 M). The lysate was centrifuged, 15,000 3 g, 20 min, and the supernatant was collected. room temperature, washed in water, and incubated in Schiff s reagent (Sigma) for 15 min at room temperature. Sections were washed and counterstained with hematoxylin. Cellprofiler software [S3] was used to quantify the number of nuclei per microscopic field in the liver of wild-type and YAP1-induced mice. Apoptosis Experiments Acute hepatocyte apoptosis was induced as described [S4]. In brief, 0.15 mg/kg of the anti-cd95 antibody (clone Jo2, BD PharMingen) was injected intraperitoneally into mice. 3 hr later, animals were killed and livers collected and fixed in 10% formalin. Apoptosis was detected by TUNEL staining with the in situ cell death detection kit (Roche) in paraffin tissue sections. In order to quantify the extent of apoptosis in the liver, four random tissue sections per animal analyzed were evaluated for TUNEL-positive cells. Electron Microscopy Samples from the small intestine were fixed in 2% gluteraldehyde, 3% paraformaldehyde, 5% sucrose in sodium cacodylate buffer (ph 7.4), washed in cacodylate buffer processed by standard procedures, and analyzed on a Philips EM410 electron microscope. Analysis of YAP1 Expression in Colon Cancer Publicly available data set of microarray expression profiles of 105 human colorectal cancers (NCBI GEO Series GSE5261) was analyzed to identify genes that are coexpressed with YAP1. Genes were ranked by Pearson correlation of log2 expression ratios, and correlation was tested with Pearson s product moment correlation coefficient, corrected for false discovery rate. BclXL exhibited the highest coexpression with YAP1 (correlation = 0.64, p < 1e-05) and cyclin D1 was ranked 18th (correlation = 0.51, p < 1e-04). Supplemental References S1. Hochedlinger, K., Yamada, Y., Beard, C., and Jaenisch, R. (2005). Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell 121, S2. Beard, C., Hochedlinger, K., Plath, K., Wutz, A., and Jaenisch, R. (2006). Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis 44, S3. Carpenter, A.E. (2007). Software opens the door to quantitative imaging. Nat. Methods 4, S4. Ogasawara, J., Watanabe-Fukunaga, R., Adachi, M., Matsuzawa, A., Kasugai, T., Kitamura, Y., Itoh, N., Suda, T., and Nagata, S. (1993). Lethal effect of the anti-fas antibody in mice. Nature 364, Histological Analysis Tissues were fixed in formalin for 24 hr and embedded in paraffin. 4 mm sections were cut and stained with hematoxylin and eosin (H&E) or used for immunohistochemical analysis. Immunohistochemical staining was done with the avidin/biotin immunoperoxidase assay (Vector Laboratories). Sections were incubated with primary antibody overnight and with secondary antibody for 1 hr. Vector Hematoxylin QS was used as nuclear counterstain. The following antibodies were used: anti-yap1, Cell Signaling, rabbit polyclonal (1:40), anti-hes1, kindly provided by T. Sudo, rabbit polyclonal (1:750), anti-ki-67, DAKO, rat monoclonal TEC-3 (1:250). Alkaline phosphatase activity stain was done with the Vector Red Alkaline Phosphatase Substrate Kit I and counterstained with hematoxylin. For periodic acid-schiff (PAS) staining, sections were deparaffinized, immersed in Periodic Acid Solution (Sigma) for 5 min at

2 S2 Figure S1. YAP1 Expression in Hepatocytes Confers Resistance to Apoptosis (A) TUNEL staining of liver sections of control or YAP-induced mice injected with PBS or 0.15 mg/kg of a monoclonal antibody against the Fas receptor. (B) Quantification of TUNEL-positive hepatocytes is shown (n = 3 animals per subgroup 6 standard deviation). (C) Liver sections from a control and LAP-YAP1 transgenic mice (induced for 5 weeks) were stained with an antibody against the mature hepatocyte marker albumin. (D) Number of nuclei per microscopic field as quantified with Cellprofiler software measurements on three independent DAPI-stained liver sections obtained from independent mice (6 standard deviation). Note that the nuclei number in the LAP-YAP1 sections is increased, indicating that hepatomegaly is due to increased cell number as opposed to increased cell size.

3 S3

4 S4 Figure S3. YAP1 Does Not Modulate OCT4 Levels (A) Western blot analysis of dysplastic intestines from R26-Oct4, R26-Yap1, and control mice, 4 days after induction. Note that OCT4 protein is undetectable after YAP1 activation, even on a long exposure. (B) Western blot analysis of R26-YAP1-inducible ES cells in the absence or presence of doxycycline. OCT4 protein levels are unaffected by YAP1 activation. Figure S2. Effects of YAP1 Activation in Epithelial Tissues (A) Ductal metaplasia of pancreatic acinar cells observed in chimeric R26-YAP1 mouse 30 days after doxycycline induction. Metaplastic lesions stain positive for YAP1 transgene and HES1. (B) Enlarged proliferative compartments in the skin of R26-YAP1 mice. After 3.5 days of induction, the skin of transgenic mice demonstrate thickening of the epidermis revealed by H&E staining (top) and an expansion of proliferating Ki67-positive cells (middle), which also express YAP (bottom).

5 S5 Figure S4. Effects of YAP1 Activation on Wnt Signaling (A) Western blot analysis of whole-cell or nuclear lysates of intestinal epithelial cells 5 days after induction, with antibodies directed against b-catenin, YAP1, and CDK2. (B) Immunohistochemical staining of EphB2 in the intestinal epithelium 4 days after YAP1 activation. (C) Time course analysis of Paneth cell localization indicates abnormal scattering of Paneth cells (arrows) along the villus region 2.5 days after YAP1 activation and absence of Paneth cells after longer induction periods. Note that Paneth cells reappear after cessation of YAP1 induction for 2 days.