1. VAP protein knockdown abolishes STARD3‐mediated cholesterol accumulation in endosomes
VAP protein knockdown abolishes STARD3‐mediated cholesterol accumulation in endosomes AWestern blot analysis of VAP‐A and VAP‐B expression in untreated (NT) HeLa/STARD3 cells or HeLa/STARD3 expressing a control shRNA (shCtrl) or two pairs of shRNAs targeting VAP‐A and VAP‐B (shVAP‐A/B‐α or shVAP‐A/B‐β). Actin was used as a loading control.BHeLa cells (a–c), and HeLa/STARD3 cells expressing a control shRNA (shCtrl; d–f) or two pairs of shRNAs targeting VAP‐A and VAP‐B [shVAP‐A/B‐α (g–i) or shVAP‐A/B‐β (j–l)] were labeled with anti‐STARD3 antibodies (magenta) and with the fluorescent cholesterol probe filipin (Cyan Hot). Merged images of filipin and STARD3 signals are shown in (c, f, i and l). Scale bars: 10 μm.CRelative fluorescence intensity of intracellular filipin signal in HeLa, HeLa/STARD3/shCtrl, HeLa/STARD3/shVAP‐A/B‐α, and HeLa/STARD3/shVAP‐A/B‐β. n = 3 independent experiments. Total number of cells analyzed: HeLa: 84; HeLa/STARD3/shCtrl: 130; HeLa/STARD3/shVAP‐A/B‐α: 109; HeLa/STARD3/shVAP‐A/B‐β: 92. Number of cells analyzed per sample per experiment ≥ 25. Mean ± SD; ***P < 0.001, ANOVA with Tukey's multiple comparison test.D–GTEM images (a) of control HeLa cells (D), HeLa/STARD3 cells (E), or HeLa/STARD3 cells expressing a control shRNA (F) or a pair of shRNAs targeting VAP‐A and VAP‐B (G). Scale bars: 200 nm. Schematic representation (b) of images shown in (a); the ER, endosomes, and intraluminal membranes are in dark, light, and median gray, respectively. Mt: mitochondria; Nc: nucleus; PM: plasma membrane.HQuantification by stereology of relative intraluminal membrane surface on TEM sections. Fifty (HeLa; HeLa/Ctrl; HeLa/STARD3) and 25 (HeLa/STARD3/shCtrl; HeLa/STARD3/shVAP‐A/B‐β) endosome sections were quantified. Mean ± SD; ***P < 0.001, Kruskal–Wallis with Dunn's multiple comparison test.
Léa P Wilhelm et al. EMBO J. 2017;embj
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