Fig. 4: TRPV4 responds to extracellular environments and regulates cell proliferation.

a Lysates of HSC-4 cells expressing control or TRPV4 #1 shRNA 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. b Fluo-4-AM-loaded HSC-4 cells expressing control or TRPV4 #1 shRNA were cultured in 96-well plates. The cells were exposed without or with 50 nM GSK1016790A, and intracellular Ca²⁺ influx was measured. c HSC-4 cells expressing control or TRPV4 #1 shRNA were cultured for 5 days in the 3D Matrigel. The cells were stained with anti-Ki-67 antibody, phalloidin and Hoechst 33342, and then Ki-67-positive cells and Hoechst 33342-stained cells were counted, and areas of spheres were calculated (n = 94). Ki-67-positive cells were counted, and results are expressed as the percentage of positively stained cells compared with total Hoechst 33342-stained cells (n = 3046). ×200 magnification, scale bars, 100 μm. d HSC-4 cells (5 × 10⁷ cells) expressing control or TRPV4 #1 shRNA were subcutaneously implanted into nude mice (n = 6). After 14 days, the nude mice were sacrificed. Representative appearance of one mouse (top picture) and extirpated xenograft tumors (bottom picture) are shown. The volumes and weights of the xenograft tumors were measured (right two graphs). Dashed lines show the outline of xenograft tumors, and arrowheads indicate the positions of tumors (top picture). Scale bars, 1 cm. e Sections prepared from xenograft tumors of HSC-4 cells were stained with anti-Ki-67 and anti-phospho-AKT antibodies and hematoxylin. Ki-67-positive cells were counted, and results are expressed as the percentage of positively stained cells compared with total hematoxylin-stained cells (n = 10,977) (right graph). Dashed lines show the outline of xenograft tumors. ×400 magnification, scale bars, 50 μm. Results are shown as means ± s.d. of three independent experiments. *P < 0.01.