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. 2015 Sep 22;11(11):2074–2088. doi: 10.1080/15548627.2015.1094597

Figure 6.

Figure 6.

Metabolic and endocrine effects of E2F1 depletion in e2f1−/− adipocyte-like cells. Confluent MEFs were exposed to differentiation medium for 14 d. At the end of differentiation period e2f1−/− and WT adipocyte-like cells were treated with inflammatory stress inducers (TNF 10 ng/ml+IL1B 10 ng/ml) for 24 h. (A) To assess insulin signaling in e2f1−/− and WT adipocyte-like cells, they were treated with inflammatory stress inducers (TNF 10 ng/ml + IL1B 10 ng/ml) for 21 h, then serum starved (in the presence of the cytokines, as indicated) for 3 h, and then incubated with insulin (0.5 and 100 nM) for 7 min to assess insulin signaling. Shown are representative blots from western blot analysis of n=3 for the p-AKT, and for the p-RPS6KB1 and p-GSK3B n=2 independent experiments yielding identical results. Densitometry of p-AKT (Ser473) to total AKT and p-RPS6KB1 to ACTB ratios are shown in graphs next to the blots (B). (C) To evaluate lipolysis in e2f1−/− and WT adipocyte-like cells, they were treated as above, medium was changed for 1 h to KRBH buffer and lipolysis was measured by evaluating glycerol and FFA release to the medium. (D) To measure secreted levels of LEP and ADIPOQ, medium was analyzed by ELISA and corrected for cell protein concentrations. Protein concentrations of cell lysates were used to normalize the results. (E) Differentiated epididymal preadipocyte cell line was electroporated with either nonspecific sequence (NS) or with siRNA targeting Atg7. Cells were then treated, as above, with TNF (10 ng/ml) + IL1B (10 ng/ml), and lysates prepared to assess the efficiency of Atg7 knockdown. Densitometry is from 5 independent experiments. (F) ADIPOQ secretion to the medium of adipocytes without or with Atg7 siRNA-mediated knockdown was assessed as described above (in D). ADIPOQ secretion to the medium of adipocytes without or with Atg7 siRNA-mediated knockdown was assessed as described above (in D). *, P<0.05; **, P<0.005; ***, P<0.005 versus con WT/NS. #, P<0.05; ###, P<0.001 versus con WT/NS. $$, P<0.005; $$$, P<0.001 versus con e2f1−/−/siAtg7. @, P<0.05 versus Ins 0.5 WT.