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 5. High Pi activates signaling pathways associated with oxidative stress in differentiated C2C12 cells. (A) Representative Nrf2 and p62 immunoblots from whole-cell lysates prepared from proliferating and differentiated C2C12 cells. β-actin was used as loading control. (B) Representative immunoblots (upper panel) and densitometric analysis (lower panel) of Nrf2, Keap1, and p62 expression in whole-cell lysates from 3-day-differentiated C2C12 cells treated for 24 h with the indicated Pi concentrations. Data are presented as means ± SEM. *P < 0.05 vs. 0 mM Pi. (C) Representative immunoblots of cytosolic and nuclear Nrf2, p62, and myogenin expression in 3-day-differentiated C2C12 cells treated for 24 h with the indicated Pi concentrations. (D) Representative confocal micrographs of Nrf2 (green), p62 (green) and myogenin (red) immunofluorescence in differentiated C2C12 cells treated with the indicated Pi concentrations. Nuclei were stained using DAPI (blue). The boxed areas within Nrf2 staining images are reproduced at higher magnification in the panels immediately below. Arrows highlight positive nuclear Nrf2 expression.