RASSF4 regulates the levels of PI(4,5)P₂. (A) Confocal images of HeLa cells expressing mCherry–PLCδ-PH and treated with either siRASSF4 or siControl. Bar, 5 µm. Relative (Rel.) mCherry–PLCδ-PH fluorescence intensity at the PM from >30 cells across three independent experiments was evaluated. Means ± SEM are shown. (B) Incorporation of myo-[³H]inositol into phosphoinositides for 24 h in HeLa cells treated with either siControl or siRASSF4. Means ± SEM of four replicates from two independent experiments are shown. PI, phosphatidylinositol. (C) Relative PM PI(4,5)P₂ levels by anti-PI(4,5)P₂ staining were quantified from >30 cells across three independent experiments. Means ± SEM are shown. (D) Confocal images of HeLa cells cotransfected with mCherry–PLCδ-PH and either RASSF4-YFP or a control vector. Bar, 5 µm. Relative mCherry–PLCδ-PH fluorescence intensity in the PM from >30 cells across three independent experiments was evaluated. Means ± SEM are shown. (E) Changes in PM PI(4,5)P₂ levels induced by 100 μM histamine (His) monitored in HeLa cells cotransfected with H1R and GFP–PLCδ-PH together with either siControl (n = 19) or siRASSF4 (n = 12). Means ± SEM are shown. (F) Changes in PM PI(4,5)P₂ levels induced by 100 μM His monitored in HeLa cells cotransfected with H1R and GFP–PLCδ-PH together with either a control vector (n = 9) or RASSF4 (n = 14). Means ± SEM are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.