MTMR 2/3/4/7 regulate the cellular level and distribution of PtdSer. (A) T47D cells stably expressing GFP-LactC2 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. Representative GFP-LactC2 images are shown. The inserted values represent a mean fraction ± SEM of CellMask colocalizing with GFP-LactC2 calculated by Manders’ coefficient from three independent experiments. Scale bar—10 μm. (B) Intact basal PM sheets prepared from T47D cells co-expressing GFP-LactC2 and shRNA targeting MTMR 2, 3, 4, or 7 were labeled with anti-GFP–conjugated gold particles and visualized by EM. The graph show a mean number of gold particles ± SEM (n ≥ 15). (C)MTMR 2/3/4/7 KD and stably expressing GFP-KRASG12V T47D cells were incubated with Cy5-conjugated annexin V, and annexin V–positive cells were counted by flow cytometry. SCR-expressing cells were treated with 1 μM STS for 6 h to induce apoptosis. The graph represents a mean (%) ± SEM of annexin V–positive cells from three independent experiments. (D) Whole-cell PtdSer levels were measured in these cells via electron spray ionization and MS/MS from three independent experiments. A heat map was constructed to quantify the changes of different lipid species after MTMR KD. Each row represents a different species of PtdSer, while each column represents a single sample. The scaled expression values of each lipid measured are plotted in a red-blue color log₂ scale. In relation to control (SCR) cells, red and blue colored tiles indicate higher and lower lipid contents, respectively. (E) The graph shows the mean of total moles ± SEM of PtdSer. Significant differences between control (SCR-expressing) and MTMR-silenced cells were assessed using one-way ANOVA tests (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, #—not significant). SCR, scrambled shRNA; STS, staurosporine.