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. 2021 Jan 6;70(3):696–709. doi: 10.2337/db20-0954

Figure 2.

Figure 2

HO1 overexpression activates NF-κB and induces HGP in hepatocytes. A: Hepatocytes isolated from wild-type mice were transfected with Adv-GFP or Adv-HO1 for 16 h and then starved in DMEM with 1% FBS for 1 h, followed by 100 nmol/L glucagon stimulation for 1 h. The cell lysates were loaded onto a 10% SDS-PAGE for Western blotting to detect HO1, pp65-S536, total p65, pp38-T180/Y182, and total p38α. The relative expression of each phosphorylated protein was normalized to the corresponding total protein and quantified by ImageJ software. n = 3/group. Results are presented as mean ± SEM. B: Hepatocytes isolated from wild-type mice were transfected with scramble siRNA or p65 siRNA for 12 h and then transfected with Adv-GFP or Adv-HO1 for 24 h. Afterward, the cells were switched to HGP buffer for 3 h. HGP was normalized to total protein levels. n = 4/group. Results are presented as mean ± SEM. C: Hepatocytes isolated from wild-type mice were pretreated by 10 µmol/L p65 inhibitor JSH23 for 0.5 h and then transfected with Adv-GFP or Adv-HO1 for 20 h, followed by incubation in HGP buffer for 3 h. HGP was normalized to total protein levels. n = 4/group. Results are presented as mean ± SEM. D: Hepatocytes isolated from wild-type mice were transfected with scramble siRNA or p65 siRNA for 12 h and then transfected with Adv-GFP or Adv-HO1 for 24 h. The cell lysates were loaded onto a 10% SDS-PAGE for Western blotting to detect HO1, total p65, pCREB-S133, total CREB, pFoxo1-S273, and total Foxo1. The relative expression of each phosphorylated protein was normalized to the corresponding total protein and quantified by ImageJ. n = 3/group. Results are presented as mean ± SEM. E: Hepatocytes isolated from wild-type mice were pretreated by 10 µmol/L p65 inhibitor JSH23 for 0.5 h and then transfected with Adv-GFP or Adv-HO1 for 24 h. The cell lysates were loaded onto a 10% SDS-PAGE for Western blotting to detect HO1, total p65, pCREB-S133, total CREB, pFoxo1-S273, and total Foxo1. The relative expression of each phosphorylated protein was normalized to the corresponding total protein and quantified by ImageJ. n = 3/group. Results are presented as mean ± SEM.