Fig. 8.

Effects of RhoGEF inhibition on U46619 and ROS-induced RhoA translocation and contraction. A-D: Fluorescence imaging of RhoA-EmGFP translocation in PASMC, co-transfected with ARHGEF1 siRNA or scrambled siRNA, stimulated with U46619 (A: U46, 100 nM) or LY83583 (C: LY, 1 µM). Quantification of spot/patch fluorescence intensity expressed as % change above control levels during stimulus ± inhibition (B: U46619, D: LY83583). ##P<0.001 vs. baseline, **P<0.001 vs. treatment, 2-way ANOVA, n=12–16 cells from a total of three different cell lines. Each measurement is combined from at least 3 spots/patches from each cell. E: Concentration-dependent relaxation responses Y16 (10 µM) in IPA pre-constricted with 100 nM U46619. Left panels: representative traces with arrows indicating where each dose was added. Right panels: mean effects of inhibitor plotted against DMSO vehicle control (n=12). *P<0.05, **P<0.01 vs. DMSO control (2-way ANOVA). F: Effects of LY83583 and Y16 on U46619-induced contraction in IPA. U46619 concentration was adjusted in each case to produce contractions equivalent to ~10% of KPSS. 1 µM LY83583 was then added for 10 min (U46+LY). This response was then repeated in the presence of Y16 (10 µM, n=13). Left panels: representative traces. Right panels: mean ± SEM measurements of peak LY83583-induced contraction, expressed as a percentage of the U46619 pre-constriction. **P<0.01 vs. U46, ##P<0.001 vs. control U46+LY (2-way ANOVA).