Extended Data Fig. 7: The recruitment of cholesterol in Sgms2-knockdown cells was restored following the addition of SM.
a, L929 cells stably expressing TSPAN4–GFP were infected for more than 24 h with lentivirus carrying non-specific-(NS) or Sgms2-shRNA (Sgms2-KD-1) constructs and then subjected to puromycin selection for 48 h. Cells were supplemented with vehicle or 5 µM SM. Cells were fixed with 4% PFA, stained with filipin III (which binds specifically to cholesterol) and observed by confocal microscopy. Scale bars, 10 µm. b, qPCR analysis of Sgms2 expression in the cells from Extended Data Fig. 7a. Sgms2-shRNA significantly reduced Sgms2 expression. Data are the mean ± s.d., n = 3 independent experiments. ****P < 0.0001; two-tailed unpaired Student’s t-test. P < 0.05 was considered statistically significant. c, Statistical analysis of summed filipin III intensity per RF length from Extended Data Fig. 7a. NS + Vehicle, n = 157 cells; NS + SM, n = 164 cells; Sgms2-KD-1 + Vehicle, n = 153 cells; Sgms2-KD-1 + SM, n = 162 cells. Data are from three independent experiments. As the data are not normally distributed, they are presented as the median with the interquartile range. ****P < 0.0001 (NS + Vehicle versus Sgms2-KD-1 + Vehicle; Sgms2-KD-1 + Vehicle versus Sgms2-KD-1 + SM); two-tailed unpaired non-parametric test (Mann–Whitney test). P < 0.05 was considered statistically significant.
