Fig. 1: TRPV4 expression in oral squamous cell carcinoma cells.

a TRPV4 mRNA levels were measured in OSCC cell lines (SAS, HSC-2, HSC-3, HSC-4, SQUU-B) using quantitative RT-PCR. Relative levels of TRPV4 mRNA expression were normalized to GAPDH and expressed as fold-changes compared with expression in MOE1a cells. b Fluo-4-AM-loaded HSC-4 cells 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 were transfected with control or two different TRPV4 siRNAs for 48 h, and TRPV4 mRNA levels were measured by quantitative RT-PCR. Relative TRPV4 mRNA levels were normalized by GAPDH and expressed as fold-changes compared with levels in control siRNA transfected cells. 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. d HSC-4 cells were transfected with control or two different TRPV4 siRNAs for 48 h and Fluo-4-AM-loaded cells were cultured in 96-well plates. The cells were exposed without or with 50 nM GSK1016790A, and intracellular Ca²⁺ influx was measured. e HSC-4 cells were transfected with control or two different TRPV4 siRNAs for 48 h and cells were loaded with 8 μM OGB-1 for last 1 h. Then, the cells were stained Hoechst 33342 and OGB-1-positive fluorescent intensity was measured. ×200 magnification, scale bars, 50 μm. Results are shown as means ± s.d. of three independent experiments. *P < 0.01.