Fig. 4. PI3Kγ is required for TRPV4 ion channel function.

(A) Representative plots showing the effects of scrambled, PI3Kα, and PI3Kγ siRNA on Ca²⁺ influx induced by the TRPV4 agonist GSK1016790A (GSK) in human lung fibroblasts. Ca²⁺ influx was measured in relative fluorescence units (RFU) using Calcium 5 dye on intact human lung fibroblast (19Lu) monolayers treated with scrambled, PI3Kα, or PI3Kγ siRNA. (B) Quantification of experiments in (A). ***P<0.005 compared with PI3Kα siRNA or scrambled siRNA. (C) Representative immunoblots showing PI3Kα and PI3Kγ in 19Lu cells treated with scrambled, PI3Kα, or PI3Kγ siRNA. GAPDH is a loading control. (D) Quantification of PI3Kα and PI3Kγ band density relative to GAPDH in (C). ***P<0.005 as indicated. (E) Representative plot showing GSK1016790A-induced Ca²⁺ influx in WT and PI3Kγ KO mouse lung fibroblasts (MLFs) treated ±TGF-β as indicated. (F) Quantification of RFU in 300 nM GSK1016790A conditions in (E). *P<0.05 as indicated. (G) Representative immunoblot showing TRPV4 in WT and PI3Kγ KO MLFs treated ±TGF-β. (H) Quantification of TRPV4 band density relative to GAPDH in (G). All graphs show means ± SEM from N = 3 independent experiments (*P≤0.05, ***P≤0.005, using ANOVA followed by Student-Newman-Keuls multiple comparisons test).