MTMR 2/3/4/7 regulate the localization of PM PI4P and ORP5. (A) WT T47D cells were infected with lentiviruses expressing SCR or shRNA targeting MTMR 2, 3, 4 or 7, followed by 1 µg/ml puromycin selection. These cells were overexpressed with GFP-P4M-SidM, GFP-PH-PLCδ1, or GFP-ORP5, fixed with 4% PFA, and imaged by confocal microscopy. Closed and open arrowheads for GFP-P4M-SidM indicate the staining of PM and Golgi complex, respectively. Closed arrowheads for GFP-ORP5 indicate the ER localization of ORP5. Scale bar—10 μm. (B and C) Intact basal PM sheets of Caco-2 cells co-expressing GFP-P4M-SidM (B) or GFP-PH-PLCδ1 (C) with shRNA targeting MTMR 2, 3, 4, or 7 were labeled with anti-GFP–conjugated gold particles and visualized by EM. The graphs show a mean number of gold particles ± SEM (n ≥ 15). Significant differences between control (SCR-expressing) and MTMR-silenced cells were assessed by one-way ANOVA tests (*P < 0.05, ****P < 0.0001, #—not significant). SCR, scrambled shRNA.