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. 2017 May 6;6(7):725–736. doi: 10.1016/j.molmet.2017.05.003

Figure 3.

Figure 3

Increase in transcription of ASK1 gene by mimickers of obesity-associated AT stresses is attenuated by absence of E2F1 in adipocytes. (A) Epididymal adipocytes were treated with several stress inducers (TNFα 10 ng/ml, FasL 1 ng/ml, GO 50 mU/ml, and Tunicamycin 5 μg/ml) for 4 or 24 h and subjected to quantitative real-time PCR analysis. The expression was normalized to β-actin and Hprt1. Results are mean ± SD of n = 5 independent experiments. ∗∗∗p < 0.001 4/24 h of each treatment vs. con. (B) Epididymal adipocytes were incubated with TNFα (10 ng/ml) or TNFα (10 ng/ml) + Act. D. (1 μg/ml) for 4 h and subjected to quantitative real-time PCR analysis. The expression was normalized to β-actin and Hprt1. Results are mean ± SD of n = 3 independent experiments. ∗∗∗p < 0.001 TNFα only versus con. ###p < 0.001 Act. D. (C, D) Epididymal adipocytes were transfected with. E2f1 siRNA (siE2f1) or non-specific siRNA (siNS) using electroporation. 24 h after transfection, adipocytes were incubated with TNFα (10 ng/ml) for an additional 24 h and analyzed using either quantitative real-time PCR or Western blot. Each transcript expression was normalized to β-actin and Hprt1 mRNA levels. β-actin was used as a loading control on the gel. Results are mean ± SD of 3 independent experiments. ∗∗∗p < 0.001 siE2f1con and TNFα vs. siNS con (middle graph), siNS TNFα vs. siNS con (right graph). ∗∗p < 0.01 siE2f1 TNFα vs. siE2F1 con. ##p < 0.01 siE2f1 TNFα vs. siNS TNFα. MEF-derived adipocyte-like cells from WT and E2F1−/− were treated with TNFα 10 ng/ml + IL-1β 10 ng/ml for 24 h and subjected to Western blot analysis. Shown are representative blots (E) and densitometry graphs (F–I) of n = 3 independent experiments. β-actin was used as a loading control. ∗p < 0.05 E2F1−/− con or WT TNFα 10 ng/ml + IL-1β 10 ng/ml vs. WT con. #p < 0.05 E2F1−/− TNFα 10 ng/ml + IL-1β 10 ng/ml vs. E2F1−/− con.