Fig. 2

HDACi upregulates Hippo pathway targets CYR61, CTGF, and TEAD luciferase reporter activity, while downregulating YAP expression. A MCF-7 and T47D cells were treated with romidepsin 50 nM for 24 h. qRT-PCR was performed for quantification of CYR61 and CTGF mRNA. Fold change (Y-axis) represents fold change of romidepsin-treated cells compared to DMSO-treated cells. Each dot represents a biological replicate (i.e., this experiment repeated 3 times). Blue: MCF-7. Red: T47D. *sign denotes p < 0.05. B A549, MCF-7, and T47D treated with romidepsin 50 nM and measured for luciferase activity by the 8xGTIIC-TEAD luciferase reporter. Activity fold changes normalized to DMSO-treated cells. Each dot represents a biological replicate (3 repeats for each cell line). (C) Heat map of mRNA expression of CTGF and CYR61 from the transcriptome datasets (see methods for full list). Abbreviations for cell lines: MCF: MCF-7 HR ( +) breast cancer cell line. T47: T47D HR ( +) breast cancer cell line. 231: MDA-MB-231 (TNBC). CUB: VM-CUB1 urothelial cancer cell line. UC: UM-UC-3 urothelial cancer cell line. SAHA: Vorinostat. siHDAC1_2: treated with combination of siRNAs against HDAC1 and HDAC2. Color palette denotes fold change compared to individual controls. D Multiple cell lines treated with romidepsin 30 nM for 24 h and blotted for YAP expression. Plus sign denotes treatment with romidepsin, minus sign denotes treatment with DMSO. (Romidepsin 30 nM was chosen for immunoblotting studies as this concentration demonstrated consistent changes across cell lines tested) E T47D cells treated with romidepsin 30 nM at various time points and blotted for YAP expression. F MCF-7 treated with romidepsin 30 nM for 24 h and blotted for YAP, phosphorylated YAP (p-YAP). G MCF-7 and T47D cells treated by romidepsin 30 nM for 24 h. qRT-PCR was performed for quantification of YAP1, TAZ, LATS1, and LATS2 mRNA. Fold change (Y-axis) represents fold change of romidepsin-treated cells compared to DMSO-treated cells. Each dot represents a biological replicate (Total 3 biological repeats). Blue: MCF-7. Red: T47D. *sign denotes p < 0.05. H Heat map of mRNA expression of TAZ and YAP from the transcriptome datasets (see methods for full list). Cell line denotation described in legend for Fig. 2C. Color palette denotes fold change compared to individual controls