Ectopic expression of WT MTMR 3 restores the effects of MTMR 3 loss. (A and B) T47D cells co-expressing MTMR 3 shRNA17 and GFP-KRASG12V (A) or GFP-LactC2 (B) were infected with lentiviruses expressing indicated mCherry-MTMR, fixed with 4% PFA, and imaged by confocal microscopy. Scale bar—10 μm. Asterisks indicate the restored PM localization of GFP-KRASG12V or GFP-LactC2. (C–F) Intact basal PM sheets were prepared from T47D cells co-expressing MTMR 3 shRNA17 and GFP-KRASG12V (C) or GFP-LactC2 (D), GFP-P4M-SidM (E), or GFP-2xFYVE (F), and infected with lentiviruses expressing indicated mCherry-MTMR members, and labeled with anti-GFP–conjugated gold particles and visualized by EM. The graphs show a mean number of gold particles ± SEM (n = 10). (G) Cell lysates from T47D cells co-expressing GFP-KRASG12V, MTMR 3 shRNA17, and mCherry-MTMR members were immunoblotted with anti-RFP antibody. Arrowheads represent the expression of indicated mCherry-MTMR members. An actin blot is shown as a loading control. Representative blots are shown from three independent experiments. Significant differences between control (MTMR 3 KD and untagged mCherry-expressing) and other cells were assessed by one-way ANOVA tests for C–F (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, #—not significant). Source data are available for this figure: SourceData F5.