MTMR 2/3/4/7 regulate the PM localization of KRASG12V. (A) T47D cells stably expressing GFP-KRASG12V were infected with lentiviruses expressing SCR or shRNA targeting MTMR 2, 3, 4 or 7, followed by 1 µg/ml puromycin selection. Cells were incubated with CellMask for 1 h at 37°C incubator, fixed with 4% PFA, and imaged by confocal microscopy. The inserted values represent a mean fraction ± SEM of CellMask colocalizing with GFP-KRASG12V calculated by Manders’ coefficient from three independent experiments. Scale bar—10 μm. (B) Intact basal PM sheets prepared from T47D cells co-expressing GFP-KRASG12V and shRNA targeting MTMR 2, 3, 4, or 7 were labeled with anti-GFP–conjugated gold particles and visualized by EM. Representative EM images are shown. Scale bar—0.1 μm. Significant differences between control (SCR-expressing) and MTMR-silenced cells were assessed by one-way ANOVA tests (*P < 0.05, ***P < 0.001, ****P < 0.0001). SCR, scrambled shRNA.