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1 Supplementary Materials for Actin cytoskeletal remodeling with protrusion formation is essential for heart regeneration in Hippo-deficient mice Yuka Morikawa, Min Zhang, Todd Heallen, John Leach, Ge Tao, Yang Xiao, Yan Bai, Wei Li, James T. Willerson, James F. Martin* The PDF file includes: *Corresponding author. Published 5 May 2015, Sci. Signal. 8, ra41 (2015) DOI: /scisignal Fig. S1. Microarray and Yap ChIP-Seq reproducibility. Fig. S2. Yap target genes are enriched in the fetal heart. Fig. S3. Yap/Tead regulatory elements are enriched in fetal heart DHS and Yap binding peaks. Fig. S4. Analysis of cell fusion in hearts 21 days after myocardial infarction. Fig. S5. Evaluation of cardiomyocyte size in Hippo-deficient hearts. Fig. S6. Cardiomyocyte protrusions in Hippo-deficient hearts. Fig. S7. Collagen gel migration assay with mitomycin C treatment. Fig. S8. Western blot analysis to quantitate sarcoglycan and syntrophin 1. Fig. S9. Scar formation in the apex resection model in mdx hearts. Fig. S10. Cardiomyocyte proliferation in the mdx apex-resected heart. Table S1. List of primers used to amplify enhancer elements and location of the Tead motif deletion.

2 Figure S1. Microarray and Yap ChIP-Seq reproducibility. Microarray was performed on P8 control and Salv CKO mouse hearts (2 biological replicates per group). Each sample has 3 technical controls. (A) Histograms show the distribution of log-transformed raw signal values which are approximate to a normal distribution. The red dotted lines indicate the mean value. (B) Quantile-Quantile plot of log2 (raw signal value) from control and Salv CKO samples. The x-axes show quantile values of standard normal distribution. The y-axes show quantile value of log-transformed signal value. (C, D) Reproducibility of microarray raw signal value in control and Salv CKO samples. Biological replicates were plotted as x-axis and y-axis values individually. Linear correlation coefficients were measured, and r is given in each diagram. The blue lines indicate the line of best fit. (E) Boxplots show that there were no significant outliers among the samples. (F) Differentially expressed genes in control and Salv CKO samples. Individual genes were plotted by using log of fold-change as the x-axis and -log10 of the p- value as the y-axis index. The differential expression threshold was fold change of at least 1.5 and P- value of at least 0.05, highlighted in blue. (G) Reproducibility of Yap ChIP-Seq (2 biological replicates). Signal intensities were measured over input sample in 5 kb bins. Pearson's correlation coefficient was calculated based on the basis of log-transformed values.

3 Figure S2. Yap target genes are enriched in the fetal heart. (A) Heat map of Yap target gene expression in human fetal and adult ventricle (N=2 biological replicates for each). The color bar represents relative expression of log-transformed value for each gene across different samples. (B) Yap target gene orthologs are more highly expressed in human fetal hearts than in adult hearts. Gene expression was measured in RPKM by using RNA-Seq profiling data. N=2 independent biological replicates. (C, D) Gene ontology analysis for genes enriched in fetal (C) (total number of genes = 700) and adult hearts (D) (total number of genes = 417). X-axes indicate Z-score of over-representation statistics; y-axes indicate percentage of gene overlay with differentially expressed genes from Salv CKO hearts. (E) Target gene expression in postnatal day 4 neonatal rat cardiomyocytes transduced with lacz or YAP1 (S127A) expressing adenovirus. Data was extracted from GSE33019, N=4 biological replicates per group. ** false discovery rate (fdr) < 0.01; * fdr < 0.05.

4 Figure S3. Yap/Tead regulatory elements are enriched in fetal heart DHS and Yap binding peaks. (A) Matched motifs in human fetal heart DHS-Seq peaks (N=11 biological replicates). Human fetal heart-specific DHS peaks were compared to human adult heart DHS peaks (N=2 biological replicates). Enriched motifs represent fetal heart-specific DHS peaks. (B) The density of enriched motifs within a 500 bp range of human fetal heart DHS peaks are shown in comparison with that of human embryonic stem cell DHS peaks (grey). (C) Heat map of enriched motifs in DHS footprints from different adult tissues and fetal compared to adult comparison. The relative enrichment was measured by the number of footprints containing each motif. (D-F) Representative genome browser tracks of Yap targets in regions of genome that are not conserved between species. DHS-Seq peaks from human fetal (green) and adult (blue) hearts and Yap ChIP-Seq peaks from mouse P8 Salv CKO hearts (black) are shown. Fgd4 contains highly conserved Tead binding elements in the promoter region (D). In Crebbp (E) and Mad2l1 (F), fetal specific DHS peaks and Yap binding peaks are located in genomic regions that are not conserved between species (non-syntenic).

5 Figure S4. Analysis of cell fusion in hearts 21 days after myocardial infarction. Left anterior descending artery occlusion (LADO) was performed with Myh6-Cre Ert ; mtmg (control) and Myh6- Cre Ert ; Salv fx/fx ; mtmg (Salv CKO) mice and hearts were collected at 21 dpmi. Three hearts were tested for each genotype. Cardiomyocyte lineage was delineated with egfp by using GFP immunostaining and non-cardiomyocyte lineage was delineated with mtomato using dsred immunostaining. Arrow shows cell fusion of two lineages detected as a double positive staining. Bars=25 µm. Right panel shows quantification of double-positive cells. Approximately 15,000 cells were counted, n.s. not significant (Mann-Whitney U test).

6 Figure S5. Evaluation of cardiomyocyte size in Hippo-deficient hearts. Left anterior descending artery occlusion or sham surgery was performed with Myh6-Cre Ert ; mtmg (control) and Myh6-Cre Ert ; Salv fx/fx ; mtmg (Salv CKO) mice and hearts were collected at 4, 10, and 21 dpmi (N=3 mice for each genotype and time point; cells measured per sample). Cells were delineated with wheat germ agglutinin (WGA, plasma membrane marker) staining and cardiomyocytes were labeled with egfp. Cross-sectional area was measured in the transverse plane. Bars are 25 µm. Statistical significance was assessed with the Mann-Whitney U test. **P>0.001.

7 Figure S6. Cardiomyocyte protrusions in Hippo-deficient hearts. (A-F) Representative images of hearts 10 days post myocardial infarction from 8 week old mice (N=3 mice per genotype). Myh6-Cre Ert ; mtmg (control, A, D) and Myh6-Cre Ert ; Salv fx/fx ; mtmg (Salv CKO, B, C, E, F). GFP and ctnt staining revealed border zone cardiomyocyte morphology. Arrowheads show protruding cardiomyocytes. (Bars= 50 µm). (G-L) P8 apex resections 4 days post resection (N=3 mice per genotype). Myh6-Cre Ert ; mtmg (control, G, H, I) and Myh6-Cre Ert ; Salv fx/fx ; mtmg (Salv CKO, J, K, L) hearts stained for ctnt and showing GFP immunofluorescence (G, J) or stained for Vinculin (H, K). Merged images showing Vinculin staining and GFP immunofluorescence are also shown (I, L).

8 (Bars=50 µm, G, J). Arrows show cells with protrusions (J). (Bars=20 µm H, I, K, L). Arrowheads show Vinculin localization in cells with protrusions (K, L). Figure S7. Collagen gel migration assay with mitomycin C treatment. Representative images of migrating cells in control and Salv sirna-treated P19 cells either untreated or pre-treated with 10 µg/ ml mitomycin C. Cells were labeled with tubulin and DNA synthesis was monitored by EdU incorporation. Bars=100 µm. Right panel shows quantitation of migrated cells. n.s. (not significant); ***P<0.01 (Mann-Whitney U test). N=3 independent experiments analyzing approximately cells per group.

9 Figure S8. Western blot analysis to quantitate sarcoglycan and syntrophin 1. (A) Western blots for the remaining two hearts from Myh6-Cre Ert ; mtmg (control) and Myh6-Cre Ert ; Salv fx/fx ; mtmg (Salv CKO) P8 mice probed with anti-sgcd (N=3 hearts total). (B) Western blot analysis for syntrophin 1 control (N=3) and Salv CKO (N=3 hearts) P8 hearts. n.s. (not significant; non-parametric Mann Whitney).

10 Figure S9. Scar formation in apex resection model in mdx hearts. Hearts from B10 control (N=11 mice) and mdx-b10 animals (N=7 mice) were resected at P1 and collected at 21 dpr. Hearts were sectioned and Trichrome staining was performed. Panels show images of the two representative biological samples from control and mdx mice. Serial sections of the hearts are shown from anterior (left) to posterior (right). Arrow point to scar formation. Bars=500 µm.

11 Figure. S10. Cardiomyocyte proliferation in mdx apex-resected heart. B6/10 control and mdx-b6/10 hearts were resected at P1 and collected 4 days post resection. EdU was injected 4 hours before harvest. (A-D) EdU staining or border zone cardiomyocytes from B6/10 control (A) and mdx-b6/10 (B) mice at 4 days post resection. Arrowheads show protrusions. (Bars=50 µm). (C, D) Higher magnification images of boxed area in (A, B). (Bars=25 µm). (E, F) Aurora B kinase staining in the border zone from B6/10 control (E) and mdx-b6/10 (F) at 4 days post resection. (Bars=25 µm). (G) Quantitation of EdU positive cells (N=3 mice for each genotype) in border zone and distal area. *p<0.05, **p<0.01, remaining column comparisons n.s (Mann-Whitney U test). (H) Aurora B kinase quantitation in B6/10 control and mdx-b6/10 hearts (N=3 mice for each genotype). *p<0.05, remaining column comparisons n.s (Mann- Whitney U test).

12 Table S1. List of primers used to amplify enhancer elements and location of the TEAD motif deletion. Name Forward primer Reverse primer Size (bp) Deletion (TEAD motif) Actrt2 CACCGGATTTGTTCTGTCT CCCACAGCTTCTAC 3205 N/A CTCATTAC CATCAA Aurkb CACCCAAGAAGAGGGTA CTACATTCCTGGGC 1218 N/A _1 GAAAGGCTAAAG TAGGTACA Aurkb _2 CACCGAGAATGGCTCAGA AGGAGAAC CACAGTTCACAGAA CAATACTGATAC (GATTC), (GGAATG) Fmn2 CACCATTCGATCTAGTCT GGGATACATAAAG CCCATCTGCCCAAA CTTCTATC (AGGAAT) Park2 CACCCGTGATCTGGCAAC GAAACTGGCGCCTT 6106 N/A TCTAC CATATTTAC Pkp4 CACCGTAAGGTGTCATAG GGTTTGAGAGGGA 3503 N/A GCTAACAGAAA ACGCATAA Ctnna 3_1 CACCAGTAGGTTGGATTA TCTGCCTGAGC GGCAAGGTATAGA GTAAGGGCTTAG (CATTCCT) Ctnna CACCCAGGATTGGACAGT GATTCAACGCATCC 4153 N/A 3_2 Enah Lin9 Sgcd GGGATTGTTTG CACCGATCAGCTTTCTCTC TCTCCCTCTC CACCCTCTTAATAGTGCC ACTCCTGTGAAC CACCATATTAGGGTCCTA CTTGTCACATAC TTTCTCTTCTCC CCTTGATCCATTTG GACTTGAGCTG CGTGTGAGACTCTA AATTGCCAGTG CTTAGGCAGCTCAT CCTCTAAC (CATTCCT), ( CATTCC, GGAATG) (AGGAATGA), (AGGAATG) (TGGAATGGG)

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