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. 2021 Sep 21;10:e62635. doi: 10.7554/eLife.62635

Figure 3. In vivo and in vitro aging are differently associated with adipose dysfunction of adipocytes differentiated from young-donor and aged-donor adipose-derived stromal cells (ASCs).

Figure 3.

The ASCs were differentiated into adipocytes for 14 days at P3, P7, and P11. (A) Cells were stained with Oil-Red-O to visualize lipid droplets 14 days post-induction, and representative micrographs are shown. (B) Quantification of Oil-Red-O staining of adipocytes differentiated from ASCs and representative scans of wells are shown. (C) Whole-cell lysates, at day 14 post-induction, of adipocytes differentiated from ASCs isolated from young and aged donors, cultured until P3, P7, and P11, were analyzed by immunoblotting. Representative immunoblots of C/EBPα, SREBP-1c, PPARγ, and tubulin (loading control) are shown. (D) Quantification of western blot was normalized to young-donor ASCs at P3. (E) Reactive oxygen species (ROS) production, normalized against 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). (F) Mitochondrial mass (normalized against DAPI) and (G) mitochondrial membrane potential were assessed in adipocytes derived from aged-donor ASCs as described in Figure 1. (H) Whole-cell lysates (extracted at day 14 post-induction) of adipocytes differentiated from young- and aged-donor ASCs and stimulated (or not) with insulin were analyzed with immunoblotting. Representative immunoblots of Akt and phospho-Akt (Ser473) are shown. (I) The phosphorylated Akt/total Akt ratio was determined in a densitometric analysis. (J) Insulin sensitivity at P11 in adipocytes differentiated from young- and aged-donor ASCs was evaluated by measuring glucose uptake in basal and insulin-stimulated conditions as described in the Materials and methods section. The insulin fold induction was determined. Results are quoted as the mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001 for aged-donor vs. young-donor ASCs, #p < 0.05, ##p < 0.01, ###p < 0.001 vs. young- or aged-donor ASCs at P3. All experiments were performed in duplicate or triplicate with ASCs isolated from four different donors in each group.

Figure 3—source data 1. Analysis by western blot of sadipogenic markers C/EBPa, SREBP1C and PPARg.