Aregs exhibit robust non-and anti-adipogenic properties that arise with age and involve RA signaling (2022)
Figure 5

Figure 5. RA signalling modulates Aregs' anti-adipogenic propertyCD142+ ASPC (Areg)-specific candidates and their involvement in adipogenic inhibition

Bulk RNA-seq-derived expression plots of Rbp1 (Retinol-binding protein 1) and Aldh1a2 (coding for Retinal dehydrogenase, RALDH2) in freshly isolated total, CD142- and CD142+ ASPCs, n=4-7, 1-5 biological replicates, 1-4 independent wells for each;

Expression heatmap listing genes linked to "retinol metabolic process" (GO:0042572) across bulk RNA-seq samples of freshly isolated CD142- and CD142+ ASPCs; genes identified as lead by GSEA (Methods) are coloured in red; log normalised expression scaled by row;

Expression heatmap of the lead genes identified by GSEA of the significantly enriched terms "cellular response to RA" (GO:0071300) and "RA receptor signalling pathway" (GO:0048384); GSEA identified these two terms as enriched when performed on the genes driving PC1 shown in Fig. 4C; see colour legend at the bottom of the figure;

Correlation plot of "RA score" (RAS) (based on the expression of genes linked to "cellular response to RA" (GO:0071300) and "RA receptor signalling pathway" (GO:0048384)) versus "CD142+ score" (Appendix Table S1) or "white fat cell differentiation score" (WFCDS) (GO:0050872);

Volcano plot showing the coefficient of the linear regression performed between "RA score" (RAS) (based on the expression of genes linked to "cellular response to RA" (GO:0071300) and "RA receptor signalling pathway" (GO:0048384)) and log normalised gene expression (RAS ~ log(normalised expression)) (x-axis) versus the -log10(p-value) (y-axis); top CD142+ markers (i.e., Areg markers, Appendix Table S1) are highlighted in blue;

Boxplots showing the distribution of the log normalised expression of F3 (top) or Mgp (bottom) across transcriptomic data of CD142- ASPCs treated with an adipogenic cocktail supplemented with DMSO or different concentrations of RA (0.01 to 100 µM); the indicated significance is based on the result of differential expression analysis between all RA treated samples or samples treated with RA of a concentration between 0.01 and 1 µM versus controls (treatment with DMI with or without DMSO), n=2-4, 2-4 biological replicates, 1 independent well for each;

Representative fluorescence microscopy images of CD142- ASPCs, co-cultured with CD142+ ASPCs (Aregs) (in the top well) subjected to the indicated compounds at the indicated concentration; AGN and BMS are antagonists of RAR, DEAB is an antagonist of RALDH enzymes which convert retinaldehyde to RA, and HX is an antagonist of RXR;

Fraction of differentiated CD142- ASPCs indicated in G, as quantified by the "adiposcore"; marker shapes correspond to different biological replicates, n=4-15, 2-5 biological replicates, 2-3 independent wells for each;

Representative fluorescence microscopy images of CD142- ASPCs, co-cultured with CD142+ ASPCs (Aregs) subjected to control (scr) or siRNA-mediated knockdowns of selected RA signalling pathway genes: Rara, Rarb, Rarg, Rbp1, and Rxrg after adipogenic differentiation (i.e., after exposure to an adipogenic cocktail, Methods);

Fraction of differentiated CD142- ASPCs indicated in I, as quantified by the "adiposcore"; marker shapes correspond to different biological replicates, n=24-29, 4 biological replicates, 5-8 independent wells for each.

Data information: In all images, nuclei are stained with Hoechst (blue) and lipids are stained with Bodipy (yellow); scale bars, 100 µm;

*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, pairwise two-sided t-test (H-J), or one-way ANOVA and Tukey HSD post hoc test (A) for statistical details see Methods.