Fig. 3: TRPV4 expression is required for cell proliferation and migration through CaMKII-mediated AKT activation.

a HSC-4 cells expressing mock or TRPV4 were transfected with control or TRPV4 #1 siRNA for 48 h, and cell lysates were probed with anti-TRPV4 and anti-β-actin antibodies. Band intensities were quantified using NIH image software and ratio of TRPV4/β-actin was expressed as fold-changes compared with control cells. Endogenous TRPV4 mRNA levels were measured by quantitative RT-PCR using primers encoding the 3’-UTR. Relative TRPV4 mRNA levels were normalized by GAPDH and expressed as fold-changes compared with levels in mock control siRNA transfected cells. b Fluo-4-AM-loaded HSC-4 cells expressing mock or TRPV4 were cultured in 96-well plates, and were exposed without or with 1, 5, 10, and 50 nM GSK1016790A. Then, intracellular Ca²⁺ influx was measured. c HSC-4 cells expressing mock or TRPV4 were transfected with control or TRPV4 #1 siRNA for 48 h. The cells were stained with anti-Ki-67 antibody and Hoechst 33342, and then Ki-67-positive cells and Hoechst 33342-stained cells were counted, respectively. Results are expressed as the percentage of Ki-67-positive cells compared with total Hoechst 33342-stained cells. ×200 magnification, scale bars, 100 μm. d HSC-4 cells expressing mock or TRPV4 were transfected with control or TRPV4 #1 siRNA were cultured in the presence of 5% FBS for the indicated numbers of days, and cell numbers were counted. e HSC-4 cells expressing mock or TRPV4 were transfected with control or TRPV4 #1 siRNA, and then the cells were placed in Transwell chamber for the migration assay. Migration activities are expressed as the percentage of control cells. ×100 magnification, scale bars, 200 μm. f HSC-4 cells expressing mock or TRPV4 were transfected with control or TRPV4 #1 siRNA for 48 h. The cell lysates were probed with anti-phospho-CaMKII and anti-pan-CaMKII antibodies (left panel) or anti-phospho-AKT and anti-pan-AKT antibodies (right panel). Band intensities were quantified using NIH image software and ratio of phospho-CaMKII/pan-CaMKII or phospho-AKT/pan-AKT was expressed as fold-changes compared with control cells. Results are shown as means ± s.d. of three independent experiments. *P < 0.01.