RASSF4 regulates the formation and stability of ER–PM junctions. (A and B, left) EM micrographs of HRP-KDEL–expressing HeLa cells treated with the indicated siRNA or plasmid for 2 d. Red arrows indicate ER–PM junctions. Bar, 2 µm. (Right) The percentage of PM length engaged in contact with the ER is shown. Means ± SEM of six to eight cells are plotted. N indicates the cell nucleus. (C and D) Densities of ER–PM junctions determined using a TIRF image series of YFP-KDEL–expressing HeLa cells that were treated with siRASSF4 or siControl (C) or transfected with RASSF4 or a control vector (D). More than 24 cells for each condition across three independent experiments were analyzed. Means ± SEM are plotted. (E) TIRF images of ER–PM junctions labeled by MAPPER in cells treated with siControl or siRASSF4. Red arrows indicate unstable junctions. Bar, 1 µm. The percentage of stable ER–PM junctions labeled by MAPPER was quantified. More than 500 puncta in six different cells for each condition were evaluated. Means ± SEM are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.