Fig. 3

LIMP-2-WT but not LIMP-2-G379W/V415W overexpression rescues cholesterol efflux from LEs in LIMP-2 deficient cells. a Principle of late endosomal BODIPY-cholesterol (BC) efflux assay in A431 cells. Cells are loaded for 24 h with oleic acid-conjugated BSA to generate lipid droplets (LD). In parallel, cells are labeled with dextran to visualize late endosomal organelles (LE). Cells are then labeled for 2 h with BODIPY cholesteryl linoleate-labeled LDL (BC LN-LDL) that enters LE, and chased in serum-free medium to monitor the transfer of BC from LE to LD (labeled with LipidTox during the last 30 min of BC-LN-LDL labeling). b Overlaid confocal images of BC (green), dextran/LIMP-2 (blue), and LipidTox (red) in live A431 cells transfected with the indicated siRNAs and/or cDNA constructs (LIMP-2-WT or LIMP-2-G379W/V415W overexpressing cells are outlined) at 0 h and 4 h of chase. Scale bar, 10 μm. c Quantification of the fraction of BC fluorescence residing in dextran-positive or LIMP-2-positive late endosomes (LE) vs. LipidTox-positive lipid droplets (LD) after 4 h of chase (n = 21 (siLIMP-2, siCtrl), 22 (siLIMP2 + WT.mCherry) or 23 (siNPC1, siLIMP-2 + G379W/V415W.mCherry) cells). Data (mean ± SEM) from 3 experiments, t-test (**P ≤ 0.01, ****P < 0.0001). d Wide-field fluorescence micrographs of filipin stained NPC1-deficient CHO M12 cells overexpressing LIMP-2-G379W/V415W-mCherry. Arrowheads indicate examples of filipin-positive LIMP-2-containing LEs. Scale bar, 10 μm. For quantification see 1 G. e Analysis of [³H]cholesterol incorporation into cholesteryl esters in LIMP-2-WT and LIMP-2-KO MEFs in the presence and absence of U18666A or PKF (n = 7 (0 h/5 h), n = 3 (9 h), n = 4 (PKF/U18666A) samples). Data (mean ± SEM) from two (9 h chase) to 3 experiments (5 h chase), unpaired two-tailed Student’s t-test (*P ≤ 0.05). Source data are provided as a Source Data file