Figure 5

TRPV4 ion channel activity and induction of CIPs. (a and b) U-2 OS cells were cultured at 37 °C or 32 °C for 24 h in the presence of indicated doses of gadolinium chloride (Gd³⁺) (a) or ruthenium red (RR) (b), and analyzed by western blot (representatives of 3 independent experiments each). (c) U-2 OS transfectants transiently expressing vector alone or shRNA against TRPV4 (shTRPV4) were cultured at 37 °C or 32 °C for 16 h, and analyzed by western blot (representative of 5 independent experiments). (d,e,f and g) U-2 OS cells were cultured at 37 °C or 32 °C for 24 h in the presence of indicated doses of RN1747 (d), GSK1016790A (e), A23187 (f), or BAPTA-AM (g), and analyzed by western blot (representatives of 3 independent experiments each). (h) Quantification of intracellular Ca²⁺ concentrations by Fura-2 in U-2 OS cells. Cells were cultured at 37 °C, 35 °C or 32 °C for 24 h in the presence or absence of 30 μM RN1734 (data indicate mean ± SEM; n = 6). Statistical significance was determined by Student’s t-test. **, P < 0.01. (i) U-2 OS cells were cultured at 37 °C or 35 °C for indicated times, and analyzed by western blot (representative of 4 independent experiments). Full-length blots are presented in Supplementary Fig. 6.