Research Paper Volume 12, Issue 21 pp 21446—21468

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.