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. Cell lysates were immunoblotted with anti-MTMR 2, 3, and 7 antibodies. Actin blots are shown as loading controls. For MTMR 4, cDNA was amplified with primers specific for MTMR 4 exons 1 and 5, or GAPDH exons 2 and 3 as a loading control. (B) T47D cells co-expressing shRNA targeting MTMR 2/3/4/7 with GFP-KRASG12V or GFP-HRASG12V were incubated with CellMask for 1 h at 37°C incubator, fixed with 4% PFA, and imaged by confocal microscopy. Representative GFP-KRASG12V and GFP-HRASG12V images are shown. The corresponding CellMask and merged images for GFP-KRASG12V are shown in Fig. S1 A. 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. (C and D) Intact basal PM sheets prepared from T47D cells co-expressing GFP-KRASG12V or GFP-HRASG12V 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 in Fig. S1 B. The graphs show a mean number of gold particles ± SEM (n ≥ 15). (E and F) Spatial mapping of the same gold-labeled PM sheets was performed. The peak values, L_max, representing the extent of KRAS and HRAS spatial organization, are shown as bar graphs (n ≥ 15). Significant differences between control (SCR-expressing) and MTMR-silenced cells were assessed by one-way ANOVA tests for B–D and bootstrap tests for E and F (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, #-not significant). SCR, scrambled shRNA. Source data are available for this figure: SourceData F1.