Research Paper Volume 12, Issue 21 pp 21446—21468

High phosphate induces skeletal muscle atrophy and suppresses myogenic differentiation by increasing oxidative stress and activating Nrf2 signaling

Figure 7. Nrf2 overexpression increases p62 and decreases myogenin promoter activity. Schematic diagram of predicted AREs (dark modules) and corresponding mutant ARE sequences within the mouse p62 (A) and myogenin (B) promoter regions, as determined using Genomatix-MatInspector software. (C, D) Luciferase reporter assay results. C2C12 cells were transiently transfected with a mSQSTM1/p62 (-2550/+63)-LUC reporter (C) or a myogenin (-2715/+52)-LUC reporter (D) and then treated for 24 h with the indicated Pi concentrations. (E, F) Luciferase activity measurements in C2C12 cells transiently co-transfected (24 h) with an Nrf2 expression plasmid plus a mSQSTM1/p62 (-2550/+63)-LUC (E) or a myogenin (-2715/+52)-LUC (F) reporter plasmid containing wild-type or mutant AREs. (G) Luciferase activity measurements in C2C12 cells transiently co-transfected with Nrf2(ARE)-LUC, mSQSTM1/p62 (-2550/+63)-LUC, or myogenin (-2715/+52)-LUC reporter plasmids plus an Nrf2 expression plasmid and treated for 24 h with the indicated Pi concentrations. (H) Representative fluorescence micrographs of C2C12 cells transfected with 0.5 μg of pEGFP.mNrf2 or pEGFP plasmid DNA in the presence or absence of 4 mM Pi. Data are presented as means ± SEM. PH, phase contrast.