Fig. 4

Functional evidence of HOTAIR regulation by EZH2. a Expression of EZH2 protein as assessed by immunoblot in the quoted non-invasive bladder cancer cell lines in parallel with HOTAIR (bar graph, assessed by RT-qPCR). b Expression of EZH2 protein (immunoblot) and HOTAIR (bar graph) in 5637 MIBC cell lines upon knockdown mediated by two different shRNA constructs. c Expression of EZH2 protein (immunoblot) and HOTAIR (bar graph) in RT112 NMIBC cell line upon transfection with CMV-EZH2-coding plasmid. d Expression of the quoted proteins and HOTAIR (bar graph) in MGH-U4 NMIBC cell line upon treatment (24 h) with NVP-BEZ35 (50 nM), rapamycin (50 nM), tyrphostin (100 μM), SB31542 (10 nM), DZNeP (10 μM), and PD98059 (10 μM). Note that HOTAIR expression is only significantly reduced upon treatment with the EZH2-specific inhibitor DZNeP. e Expression of EZH2 protein (immunoblot) and HOTAIR (bar graph) in MGH-U4 NMIBC cell line upon treatment for different time periods with DZNeP (10 μM). GAPDH and ACTIN were used for loading control in immunoblots, and TBP was used as a normalizer gene for RT-qPCR