Aregs exhibit robust non-and anti-adipogenic properties that arise with age and involve RA signaling (2022)
Figure 3

Figure 3. CD142+ ASPC (Areg)-specific candidates and their involvement in adipogenic inhibition

Representative fluorescence microscopy images of CD142- ASPCs co-cultured with an empty transwell (T-well) or with wild-type (WT) CD142+ ASPCs after in vitro adipogenic differentiation;

Fraction of differentiated CD142- ASPCs co-cultured with an empty transwell (T-well) or with wild-type CD142+ ASPCs (non-treated or treated with control scrambled siRNA) after in vitro adipogenic differentiation and as quantified by the "adiposcore"; marker shapes correspond to different biological replicates, n=10, 4 biological replicates, 2-3 independent wells for each;

Mean adipocytes size or number of quantified adipocytes in matrigel plugs which were implanted into the subcutaneous adipose depot after three weeks of high-fat diet. The plugs were injected with either only CD142- ASPCs, or with a mix containing 10-20% of CD142+ ASPCs. Markers correspond to different biological replicates, n=12-36, 1-3 biological replicates (6 mice in total), 9-12 slides for each;

Representative histology cuts of matrigel implants after fixation with 4% PFA and dehydration of plugs injected with CD142- ASPCs (top) or 80% of CD142- ASPCs and 20% of CD142+ ASPCs (bottom). Both images are plugs dissected from a female mouse after matrigel injection and 3 weeks on high-fat diet. In pink: matrigel, in white: adipocytes, scale bar = 500µm, staining H&E Fat;

Expression heatmap listing genes linked to "negative regulation of fat cell differentiation" (GO:0045599) across bulk RNA-seq samples of CD142+ and CD142- ASPCs after adipogenic differentiation; the genes are ordered from top to bottom by the log2FC of CD142+ over CD142- ASPCs after adipogenic differentiation; significantly differentially expressed genes (FDR < 0.05) are coloured in red; log normalised expression scaled by row; T-well - transwell, scr - scrambled siRNA (control);

Representative fluorescence microscopy imaged of human-derived stromal vascular fraction (SVF), containing the ASPCs, after in vitro adipogenic differentiation when co-cultured with total, CD142- or CD142+ mouse ASPCs;

Fraction of differentiated human-derived SVF, containing the ASPCs co-cultured with total, CD142- or CD142+ mouse ASPCs after in vitro adipogenic differentiation (Methods) shown in E; marker shapes correspond to different biological replicates, n=9, 3 biological replicates, 3 independent wells for each;

Bulk RNA-seq-derived expression plots of CD142+ ASPC (Areg) markers coding for secreted proteins that were selected for downstream validation: F3 (coding for CD142), Mgp (coding for Matrix Gla protein, MGP), Gdf10 (GDF10), Clec11a (CLEC11A), Cpe (Carboxypeptidase E, CPE) and Bgn (Biglycan, BGN), n=4-7, 1-5 biological replicates, 1-4 independent wells for each;

Fraction of differentiated adult-derived CD142- ASPCs, as quantified by the "adiposcore", treated with the indicated recombinant proteins shown in J; marker shapes correspond to different biological replicates, n=11-59, 2-10 biological replicates, 3-9 independent wells for each;

Representative fluorescence microscopy images of adult-derived CD142- ASPCs after in vitro adipogenic differentiation; the induction cocktail was supplemented with recombinant proteins corresponding to the selected Areg-specific candidates: CD142, MGP, GDF10, CLEC11A, CPE and BGN at 100 ng/ml (Methods).

Data information: In all images, nuclei are stained with Hoechst (blue) and lipids are stained with Bodipy (yellow); scale bars, 100 µm, bar colours: total ASPCs - brown, CD142- ASPCs - yellow, CD142+ ASPCs (Aregs) - blue, recombinant BSA or DMSO treatment (negative controls) - light grey, recombinant EGF treatment (positive control) - dark grey;

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