Fig. 2: S33A mutant cells exhibit larger lipid droplets with reduced lipolysis and fatty acid oxidation. | Nature Communications

Fig. 2: S33A mutant cells exhibit larger lipid droplets with reduced lipolysis and fatty acid oxidation.

From: Phosphorylation of 17β-hydroxysteroid dehydrogenase 13 at serine 33 attenuates nonalcoholic fatty liver disease in mice

Fig. 2

a Huh7 cells were infected with lentiviruses carrying GFP, GFP-tagged 17β-HSD13 WT or GFP-tagged S33A mutant. Oil red O staining (scale bar, 50 μm) and confocal images (scale bar, 5 μm) were analyzed. Lipid droplets were stained by Nile red (red) and the nucleus was stained by DAPI (blue). LD size (b) and TG content (c) were analyzed. GFP: n = 4; WT: n  =  4; S33A: n  =  4 biologically independent cells. d N-SIM-3D reconstruction of the subcellular localization of 17β-HSD13 WT and 17β-HSD13 S33A protein in Huh7 cells. Lipid droplets were stained with Nile red (red) and 17β-HSD13 WT and 17β-HSD13 S33A proteins were visualized by green fluoresce. e, f Huh7 cells were stably infected with the lentiviruses carrying a full-length GFP (GFP), GFP-tagged 17β-HSD13 WT (WT) or GFP-tagged 17β-HSD13 S33A mutant (S33A). Mitochondrial respiration was analyzed in real-time using the Seahorse XF24 Extracellular Flux Analyzer. The oxygen consumption rate (OCR) (e) at different stages of respiration, basal and spare respiratory capacity (f) were measured as described in the “Methods” section. Data presented are the mean ± SEM. One-way ANOVA with Bonferroni post hoc analysis was performed. GFP: n = 5; WT: n = 4; S33A: n = 4 biologically independent cells. g–i Lipolysis was induced by forskolin treatment (10 μM) for 24 h in Huh7 cells with stable expression of GFP, 17β-HSD13 WT (WT) or GFP-tagged 17β-HSD13 S33A mutant (S33A). Oil red O staining was performed (g) and the LD sizes (h) were analyzed. i Glycerol levels in culture medium were measured. GFP: n = 5; WT: n = 4; S33A: n = 4 biologically independent cells. j–l Oxygen consumption rate (OCR) in the presence of exogenous palmitic acid in WT and the S33A mutant Huh7 cells. Advanced ORC was measured in real-time using the Seahorse® metabolic flux analyzer as described in the section of “Methods”. j The OCR curves. The times of addition of etomoxir (ETO), oligomycin (oligo), FCCP, and rotenone + antimycin A (Rot/AA) were indicated at the top. The OCRs in the presence of palmitate-BSA (PA) and ETO were displayed. k Acute response due to the addition of ETO under basal condition. l Maximal respiration due to the addition of FCCP. Medium-WT-PA: n = 5; Medium-S33A-PA: n = 4; ETO-WT-PA: n = 5; ETO-S33A-PA: n = 4 biologically independent cells. Data represent mean ± SEM; Two-tailed student’s t test was performed for b, c; One-way ANOVA with Bonferroni post hoc analysis was performed for e–l.

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