MOSPD2 is involved in LD homeostasis. (A) Western blot analysis of MOSPD2 protein level in control HeLa cells (WT), HeLa cells transfected with control siRNAs (siCtrl), siRNAs targeting MOSPD2 (siMOSPD2), and in two MOSPD2-deficient HeLa cell clones (KO#1 and KO#2) established by CRISPR/Cas9 gene editing. (B) Representative confocal images of parental HeLa cells (WT), of HeLa cells transfected with control siRNAs (siCtrl), and with siRNAs targeting MOSPD2 (siMOSPD2), and of MOSPD2-deficient HeLa cell clones (KO#1 and KO#2) labeled with BODIPY 493/503 (LDs, magenta) and Hoechst 33258 (nuclei, blue). Scale bars: 10 µm. Images were acquired on a spinning-disk confocal microscope (Nikon CSU-X1, 100× NA 1.4). The contour of each cell is delimited by a white dotted line. (C) Number (a) and area (b) of LDs in cells shown in B. Data are displayed as Superplots (Lord et al., 2020) showing the mean number and area of LDs per cell (small dots) or per independent experiment (large dots). Independent experiments (n = 5) are color-coded. Means and error bars (SD) are shown as black bars. Data were collected from 398 (WT), 323 (siCtrl), 280 (siMOSPD2), 333 (KO#1), and 413 (KO#2) cells. One-way ANOVA with Tukey’s multiple comparisons test (ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n = 5 independent experiments). (D) a: TLC analysis of lipids extracted from cells shown in A. Neutral lipids were separated with hexane/diethylether/AcOH (80:20:2 vol/vol) and revealed with primuline. CE and TAG were used as standards. b and c: Relative levels of CE (b) and TAG (c) detected by TLC. Means and error bars (SD) are shown. One-way ANOVA with Tukey’s multiple comparisons test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 4 independent experiments). (E) Enzymatic quantification of cholesterol (a), cholesterol ester (b), triacylglycerol, (c) and phospholipid (d) in control HeLa cells (WT) and MOSPD2-deficient HeLa cell clones (KO#1 and KO#2). Means and error bars (SD) are shown. One-way ANOVA with Tukey’s multiple comparisons test (*, P < 0.05; **, P < 0.01; n = 6 independent experiments). A.U., arbitrary unit. Source data are available for this figure: SourceData F2.
Figure 2.

MOSPD2 is involved in LD homeostasis. (A) Western blot analysis of MOSPD2 protein level in control HeLa cells (WT), HeLa cells transfected with control siRNAs (siCtrl), siRNAs targeting MOSPD2 (siMOSPD2), and in two MOSPD2-deficient HeLa cell clones (KO#1 and KO#2) established by CRISPR/Cas9 gene editing. (B) Representative confocal images of parental HeLa cells (WT), of HeLa cells transfected with control siRNAs (siCtrl), and with siRNAs targeting MOSPD2 (siMOSPD2), and of MOSPD2-deficient HeLa cell clones (KO#1 and KO#2) labeled with BODIPY 493/503 (LDs, magenta) and Hoechst 33258 (nuclei, blue). Scale bars: 10 µm. Images were acquired on a spinning-disk confocal microscope (Nikon CSU-X1, 100× NA 1.4). The contour of each cell is delimited by a white dotted line. (C) Number (a) and area (b) of LDs in cells shown in B. Data are displayed as Superplots (Lord et al., 2020) showing the mean number and area of LDs per cell (small dots) or per independent experiment (large dots). Independent experiments (n = 5) are color-coded. Means and error bars (SD) are shown as black bars. Data were collected from 398 (WT), 323 (siCtrl), 280 (siMOSPD2), 333 (KO#1), and 413 (KO#2) cells. One-way ANOVA with Tukey’s multiple comparisons test (ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n = 5 independent experiments). (D) a: TLC analysis of lipids extracted from cells shown in A. Neutral lipids were separated with hexane/diethylether/AcOH (80:20:2 vol/vol) and revealed with primuline. CE and TAG were used as standards. b and c: Relative levels of CE (b) and TAG (c) detected by TLC. Means and error bars (SD) are shown. One-way ANOVA with Tukey’s multiple comparisons test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 4 independent experiments). (E) Enzymatic quantification of cholesterol (a), cholesterol ester (b), triacylglycerol, (c) and phospholipid (d) in control HeLa cells (WT) and MOSPD2-deficient HeLa cell clones (KO#1 and KO#2). Means and error bars (SD) are shown. One-way ANOVA with Tukey’s multiple comparisons test (*, P < 0.05; **, P < 0.01; n = 6 independent experiments). A.U., arbitrary unit. Source data are available for this figure: SourceData F2.

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