RASSF4 regulates the levels of PI(4)P and PIP5Ks at the PM. (A) Confocal images of HeLa cells expressing N-PH-ORP5-GFP and treated with either siControl of siRASSF4. Bar, 5 µm. Relative N-PH-ORP5-GFP fluorescence intensity in the PM from >30 cells across three independent experiments was evaluated. Means ± SEM are shown. (B) Relative PM PI(4)P levels were detected by anti-PI(4)P antibody in HeLa cells treated with either siControl or siRASSF4 from ∼30 cells across three independent experiments. Means ± SEM are shown. (C) Confocal images of HeLa cells expressing YFP-PIP5K1B, treated with either siRASSF4 or siControl. Bar, 5 µm. Relative YFP-PIP5K1B fluorescence intensity in the PM from >30 cells across two independent experiments was evaluated. Means ± SEM are shown. (D) A diagram of the rapamycin-inducible approach that selectively increases PI(4,5)P₂ at the PM. (E) Relative PM PI(4,5)P₂ levels in HeLa cells transfected with the indicated siRNA, Lyn-FRB, and either CFP-FRBP-PIP5K or CFP-FRBP-PIP5Ki (control) were monitored by PI(4,5)P₂ immunostaining. More than 30 cells for each condition across three independent experiments were evaluated. Means ± SEM are plotted. (C–E) Cells were treated with 5 µM rapamycin for 10 min after the transfection and before the experiments. (F) The density of stable ER puncta in HeLa cells treated with the indicated siRNA and expressing YFP-KDEL, mCherry-KRAS-tail, Lyn-FRB, and either CFP-FRBP-PIP5K or CFP-FRBP-PIP5Ki. More than 15 cells for each condition across two independent experiments were analyzed. Plotted are means ± SEM. (G) HeLa cells were transfected with the indicated siRNA, Lyn-FRB, and either CFP-FRBP-PIP5K or CFP-FRBP-PIP5Ki. Cells were stimulated with 100 µM His, 1 µM TG, and 2 mM EGTA; 2 mM Ca²⁺ was added 6 min after stimulation. Mean peak values of Fura-2 ratio ± SEM of the Ca²⁺ add-back phase are plotted from >300 cells for each condition across three independent experiments. **, P < 0.01; ***, P < 0.001. Rel., relative.