FIGURE 1.

TM4SF5 localized at MLCSs in hepatocytes. (A) Quantification of MLCSs/cell from SNU449 hepatocytes transfected with mito‐GFP plus HA‐empty vector (EV) or HA‐TM4SF5 and treated with lysotracker. Images were analyzed for mitochondria‐lysosome co‐stains over total lysosome stains. Twenty‐nine EV and 15 TM4SF5 cells were analyzed and combined from three independent experiment. Data were represented as mean ± standard deviation (SD). ^** P < 0.01; two‐tailed unpaired t test. (B) Quantification of MLCSs from Huh7 cells stably transfected with mito‐GFP plus non‐specific shRNA (shNS, n = 43), shTM4SF5 #4 (n = 40), or shTM4SF5 #12 (n = 49) and labelled with lysotracker. Cells in normal culture media were analyzed from 3 independent experiment. ^* P < 0.05; ordinary one‐way ANOVA. (C) SNU449 cells transfected with mCherry‐TM4SF5 (red) and mito‐GFP (green) were processed for confocal microscopy with LAMP1 immunostaining (blue) (Insert “a” and “b”). Fluorescence intensities over the (“a”) and (“b”) lines were measured and graphed. (D) SNU449 cells transfected with APEX2‐TM4SF5 were glucose starved for 16 h and then replete with 10 mmol/L glucose for 30 min, before processing to DAB staining and electron microscopy. M, mitochondria; T, TM4SF5 stains on lysosome; yellow arrow, mitochondria‐lysosome contact site. Scale bar = 1 μm. (E‐F) SNU449 cells transfected with mCherry‐TM4SF5 (red) and mito‐GFP (green) were imaged for LAMP1 fluorescence (blue) using an Airyscan microscope (E) or confocal live imaging (F). Data represent 3 independent experiments. See also Supplementary Figure S1. APEX2‐TM4SF5, TM4SF5 tagged with ascorbate peroxidase 2 (APEX2); DAB, diaminobenzidine, LAMP1, lysosomal‐associated membrane protein 1; EV, empty vector; mito‐GFP, mitochondria‐targeting sequence of COX4 tagged with green fluorescent protein gene; MLCS, mitochondria‐lysosome contact site; shNS, shRNA against non‐specific sequence; shTM4SF5, shRNA against TM4SF5 sequences (of #4 or #12).