Fig. 7: GPNMB-mutation facilitated lysosome-mediated lipolysis in macrophages.

A Representative images of WT BMDMs and Gpnmb^R150X BMDMs treated with OxLDL for 24 h, followed by the staining of Lyso-Tracker Red (LTR, Red) and BODIPY staining (Green), N = 5 independent experiments, Scale bar, 5 μm. B, Representative living cell images of FLCN expression (Green) in WT BMDMs and Gpnmb^R150X BMDMs treated with CF647-OxLDL (Magenta) for 36 h, N = 5 independent experiments, Scale bar, 2 μm. C The localization of BODIPY-labeled LDs (left) or Dil-OxLDL (right) with lysosomes quantified with PCC (mean ± SEM, n = 5/4/7/10/8/10 in the left figure, n = 5/5/9/9 in the right figure). D Representative images of WT BMDMs and Gpnmb^R150X BMDMs stained with LTR-labeled lysosomes (Red) (Scale bar, 10 μm) and the quantitative analysis of LTR intensity (mean ± SEM, N = 5 independent experiments, n = 42 images each group). E Representative images of WT BMDMs and Gpnmb^R150X BMDMs stained with acridine orange (AO) (Scale bar, 5 μm) and the quantitative analysis of intensity per cell (mean ± SEM, N = 3 independent experiments, n = 9 images each group). F Representative images of WT BMDMs and Gpnmb^R150X BMDMs stained with LysoSensor Yellow/Blue DND-160 (PDMPO) (Scale bar, 10μm) and the quantitative analysis of probe intensity ratio (mean ± SEM, N = 3 independent experiments, n = 16 images each group). G Representative images of WT BMDMs and Gpnmb^R150X BMDMs transfected with R2pH-LAMP1-3×GLAG plasmid (Scale bar, 10 μm) and the quantitative analysis of intensity ratio (mean ± SEM, N = 3 independent experiments, n = 13 or 11 images). H Representative images of AO-stained BMDMs from WT ApoE^−/−, HET ApoE^−/−, and Gpnmb^R150X ApoE^−/− mice (captured with a high-content microscope, Scale bar, 500 μm) and corresponding quantitative analysis of intensity ratio (mean ± SEM, N = 3 independent experiments, n = 9 images each group). I Representative images of LTR-labeled lysosomes (Red) in WT BMDMs transfected with pEGFP-GPNMB plasmid or empty vector (Scale bar, 10 μm) and the quantitative analysis of LTR intensity (mean ± SEM, N = 3 independent experiments, n = 8 images each group). J Representative images of LysoSensor Yellow/Blue DND-160 (PDMPO) staining in WT BMDMs transfected with pEGFP-GPNMB plasmid or empty vector (Scale bar, 10 μm) and the quantitative analysis of probe intensity ratio (mean ± SEM, N = 3 independent experiments, n = 11 or 7 images). K Heatmap of the expression of lysosome-associated genes and ATP6V family genes from RNA-seq data of WT BMDMs and Gpnmb^R150X BMDMs treated with or without OxLDL. L Representative images from immunofluorescence of GPNMB expression (Red) and ATP1A1 + ATP1A2 + ATP1A3 + ATP1A4 expression (Green, upper) or ATP6V0E2 expression (Green, lower) in WT BMDMs, N = 3 independent experiments. M Representative western blot images showing the expression of GPNMB and LAMP1 in BMDMs from WT, HET, and Gpnmb^R150X mice treated with or without OxLDL (upper) and the quantitative analysis (lower) (mean ± SEM, n = 3). N Force-directed network visualization of the STRING links between enriched proteins related to the V-ATPase (c) from whole WT BMDM lysates using an anti-GPNMB antibody. Data are expressed as mean ± SEM. P values were calculated by unpaired Student t-test (C–G, I, J) or one-way ANOVA (H, M). Differences are significant for *P  <  0.05, **P  <  0.01, ***P  <  0.001, ****P  <  0.0001.