Fig. 5.

StARD3 contributes to late endosome-cholesterol egress and neutral lipid accumulation in AnxA6-deficient NPC1 mutant cells. a CHO M12-A6ko cells expressing control siRNA (siCtrl) or siRNA targeting StARD3 (siStARD3) were starved in 5% LPDS for 48 h before loading with 50 µg/ml LDL for 24 h. Cells were fixed and stained with filipin (cholesterol, red) and BODIPY 493/503 (neutral lipids, green). Enlarged regions of interest are shown. For better comparison of filipin and BODIPY staining, the outline and shape of cells is indicated. Scale bar, 10 μm. b Western blot analysis (StARD3, tubulin) and RNA quantification determined by qPCR (n = 3) of StARD3 knockdown in CHO M12-A6ko cells is shown. c–d Dot-plot of number, area and relative intensity of filipin-stained (LEs) and BODIPY-stained (LDs) vesicles per cell of a representative experiment (n > 60, 3 experiments). For quantification details see “Materials and methods”. e CHO M12 cells expressing control siRNA (siCtrl) or siRNA targeting TBC1D15 and StARD3 (siTBC1D15 siStARD3) were starved in 5% LPDS for 48 h and loaded with 50 µg/ml LDL for 24 h. Then cells were fixed, stained with filipin (cholesterol, red) and BODIPY 493/503 (neutral lipids, green), and representative fields (merged and split channels) are shown. Enlarged regions of interest are shown. For better comparison of filipin and BODIPY staining, the outline and shape of cells is indicated. Scale bar, 10 μm. f Western blot analysis of TBC1D15, StARD3 and actin ± siRNA-mediated TBC1D15 and StARD3 knockdown from lysates of CHO M12 cells as indicated. g–h Dot-plot of number, area and relative intensity of filipin-stained (LE) and BODIPY-stained (LD) vesicles per cell of a representative experiment in CHO M12 cells ± siTBC1D15 and siStARD3 as indicated (n > 60, 3 experiments). For quantification details see “Materials and methods”.**p < 0.01; ***p < 0.001 by two-tailed Student’s t test (b–d, g, h). Data are shown as mean ± SEM (b) or as mean ± SD in red (c, d, g, h)