Figure 7.

mTOR mediates PGC-1β induction through alternative NF-κB. (A) Whole-cell extracts were prepared from C2C12 cells under proliferating (myoblast) or differentiated (myotube) conditions, and Western blotting was performed (ns denotes a nonspecific band). (B) C2C12 myotubes were treated with rapamycin for 24 h, and PGC-1β levels were monitored. ChIP was performed for the methylated H3K4 (meH3K4) chromatin mark in the PGC-1β promoter. (C) Cell extracts were prepared as in A. mTOR or IgG as a control was individually immunoprecipitated (IP), and lysates were subsequently probed (IB, immunoblot) for IKKα. (D) C2C12 cell extracts from myoblasts or myotubes were prepared, and IKK kinase activity (KA) was assessed in the absence (−) or presence (+) of 20 nM rapamycin. (E) Myoblasts were transfected with the κB-S2–luciferase reporter, and after differentiation and treatment, cells were harvested for luciferase activity. (F) C2C12 myoblasts were differentiated, and ChIP analysis for endogenous RelB binding to the κB-S2 site of PGC-1β was performed in the absence or presence of 20 nM rapamycin. (G) C2C12 myoblasts were transfected with RelB, and after differentiation, cells were either untreated (−) or treated (+) with rapamycin and subsequently monitored for PGC-1β by real-time RT-PCR. Asterisks denote significance. Error bars are means ± SEM. CMV, cytomegalovirus; RLU, relative luciferase unit.