Figure 3.

Inhibition of YAP/TAZ Enhances Cardiac Reprogramming and Suppresses Fibroblast Signature

(A and B) FACS analysis of cTnT expression after 1 week. GHMT-transduced fibroblasts cultured on 126 kPa hydrogels were transduced with Scramble shRNA and YAP and TAZ-specific shRNA (shYAP and shTAZ). (B) Quantitative data; n = 3 independent triplicate experiments.

(C and D) Immunocytochemistry of cTnT and DAPI after 4 weeks. Cells were treated as described in (A). High-magnification views, as insets, show sarcomeric organization. (D) Quantitative data; n = 3 independent triplicate experiments.

(E) Quantitative data for the number of spontaneously beating cells after 4 weeks; n = 3 independent triplicate experiments.

(F) Heatmap of microarray data illustrating the global gene expression pattern of fibroblasts 2 weeks after transduction with GHMT/scramble shRNA, GHMT/shYAP, or GHMT/shTAZ cultured on 126 kPa hydrogels. Differentially expressed genes are shown; n = 2 independent biological replicates.

(G and H) Venn diagram showing genes upregulated after GHMT/shYAP and GHMT/shTAZ transduction by more than 2-fold compared with that of GHMT/scramble shRNA. (H) GO analysis of the 135 upregulated genes of the GHMT/shYAP and GHMT/shTAZ groups. Cardiac-related GO terms are shown.

(I and J) Venn diagram showing genes downregulated after GHMT/shYAP and GHMT/shTAZ transduction by more than 2-fold compared with that of GHMT/scramble shRNA. (J) GO analysis of the 472 downregulated genes in both GHMT/shYAP and GHMT/shTAZ groups. Fibroblast-related GO terms are shown.

(K and L) qRT-PCR analysis to determine relative mRNA expression of cardiomyocyte, fibroblast, and YAP/TAZ target genes in the (K) GHMT/shYAP and (L) GHMT/shTAZ groups compared with GHMT/scramble shRNA groups; n = 3 independent triplicate experiments.

All experiments were performed on 126 kPa hydrogels. All data are presented as the means ± SD. ^∗p < 0.05 versus the relevant control. Scale bars represent 100 μm.