MTMR 2/3/4/7 regulate PM PI3P contents. (A) T47D cells expressing SCR or shRNA targeting MTMR 2, 3, 4 or 7, followed by 1 µg/ml puromycin selection, were overexpressed with GFP-2xFYVE. Cells were incubated with CellMask for 1 h at 37°C incubator, fixed with 4% PFA, and imaged by confocal microscopy. Representative GFP-2xFYVE images are shown. Their corresponding CellMask and merged images are shown in Fig. S3. A selected region (the white square) is shown at a higher magnification. Arrowheads indicate the PM staining of GFP-2xFYVE. (B) The graph represents a mean fraction ± SEM (n = 13) of CellMask colocalizing with GFP-2xFYVE calculated by Manders’ coefficient. (C) Acidic lipids were extracted from MTMR 2/3/4/7 KD T47D cells, and total PI3P was measured by a quantitative ELISA. The graph shows a relative mean total PI3P level ± SEM from three independent experiments. (D and E) Intact basal PM sheets prepared from these T47D cells from A (D), or WT T47D cells expressing GFP-MTMR members (E) were labeled with anti-GFP–conjugated gold particles and visualized by EM. The graphs show a mean number of gold particles ± SEM (n ≥ 15). (F) Cells from E were fixed with 4% PFA and imaged by confocal microscopy. Arrowheads indicate the PM localization of GFP-MTMR members. Scale bar—10 μm. Significant differences between control (SCR-expressing) and MTMR-silenced cells were assessed by one-way ANOVA tests for B–D (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, #–not significant). Significant differences between untagged GFP (control) and GFP-MTMR–expressing cells were assessed by one-way ANOVA test for E (**P < 0.01, ***P < 0.001, ****P < 0.0001). SCR, scrambled shRNA.