Figure 4. High Pi induces ROS generation in differentiated C2C12 cells. (A) Analysis of cytosolic ROS levels via H2DCFDA flow cytometry in proliferating (10% FBS) and differentiated (2% HS) C2C12 cells treated with the indicated concentrations of Pi for 24 h. (B) Bar graph summarizing the data from panel A. Cells exposed to 1 mM H2O2 served as positive control. *P < 0.05, **P < 0.01 vs. 0 mM Pi. (C) Assessment of mitochondrial and cytosolic ROS levels via MitoSOX Red and H2DCFDA flow cytometry. Differentiated C2C12 cells were treated for 24 h with 4 mM Pi plus the mitochondria-targeted ROS scavenger Mito-TEMPO (10 μM) or the cytosolic ROS scavenger NAC (10 mM). (D) Bar graph summarizing the data from panel (C). *P < 0.05, **P < 0.01, ***P < 0.001. (E) Representative immunoblot and (F) densitometric analyses of protein synthesis (mTOR and S6K) and degradation (MuRF1 and atrogin-1) markers in 3-day-differentiated C2C12 cells treated for 24 h with the indicated concentrations of Pi. *P < 0.05, **P < 0.01, ***P < 0.001 vs. 0 mM Pi. #P > 0.05. Data are presented as means ± SEM.