Extended Data Fig. 10: PAQR4 promotes ceramide synthase stability.
From: PAQR4 regulates adipocyte function and systemic metabolic health by mediating ceramide levels
(a) Cers expressions during SVF-derived adipocyte differentiation (Day 4 group, n = 6; other groups, n = 4). (b) Cers expressions in sWAT of Paqr4ad and WT mice fed dox chow for 2 weeks (WT, n = 12; Paqr4ad, n = 18). (c) Representative staining of ER marker KDEL and CERS2 in sWAT and gWAT from 2 weeks of dox chow-fed Paqr4ad and WT mice (n = 3). Scale bar 50 µm. (d) Quantification analysis of Myc-CERS2 (C2, normalized by β-Tubulin) during the treatment of MG132 or bafilomycin A1 (BFA) (n = 3). (e) PAQR4 prevents CERS2 lysosomal degradation. HEK293A cells were transfected with Halo-Cers2 and SNAP-Paqr4 vectors for 24 h, and then treated with BFA for 8 h. CERS2 was labelled with HaloTag TMR ligand (red) and PAQR4 was labelled with SNAP-Cell Oregon Green (green). Cells were then fixed and stained with lysosomal marker LAMP1. Scale bar, 50 μm. Representative images from 2 independent assays with similar results. (f-g) ‘Pulse–chase’ indicates PAQR4 stabilization of CERS2. CERS2 or PAQR4 were first labelled with HaloTag TMR ligand (red) or SNAP-Cell Oregon Green (green), respectively, cells were then imaged at the indicated time points. About 20 cells of each condition were analysed. (n = 3 biological samples for each group.) Scale bar 10 μm. (h) Effect of PAQR4 on CERS5 protein levels in the presence of cycloheximide (CHX), MG132, or BFA (n = 2). (i) PAQR4 prevents CERS5 lysosomal degradation in HEK293A cells. Cells were transfected with Halo-Cers5 and SNAP-Paqr4 vectors for 24 h, and then treated as in (e). Representative images from 2 independent assays with similar results. Scale bar 50 μm. Data shown as mean ± SEM and analysed by two-way ANOVA followed by Holm-Sidak multiple-comparison test (a, g) and two-tailed unpaired t-test (b).
