Anti p akt

Primary muscle cultures (pp6) were isolated as described (Qu et al., 1998 (link)) from the indicated mouse lines. FoxO3-depleted (FoxO-KO) pp6 cells were prepared from foxO3^+/− mice as previously described (Dentice et al., 2010 (link)). C2C12 cells were obtained from ATCC. In some experiments, endogenous T3 and T4 were removed from the FBS by charcoal absorption (Larsen, 1972 (link)). Transient transfections were performed using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. Single myofibers were prepared from the extensor digitorumlongus and gastrocnemius muscles of 6- to 12-week-old mice as previously described (Rosenblatt et al., 1995 (link)). Pax7^Hi-Lo isolation by FACS has been described elsewhere (Rocheteau et al., 2012 (link)). Anti-MyoD (sc-304), myogenin (sc-12732), tubulin (sc-8035), and anti-FoxO3 antibodies were purchased from Santa Cruz Biotechnology. Polyclonal anti-MHC antibody (MF-20a) and anti-Pax7 antibody were from Developmental Studies Hybridoma Bank. Anti-D3 antibody is described elsewhere (Huang et al., 2003 (link)). Anti-Desmin antibody was from MP Biomedicals (#10519). Anti-total Akt was from Upstate, and anti-pAkt and anti-PARP were from Cell Signaling Technology. TUNEL assay was performed using the ApopTag kit from Millipore. Caspase-3/Caspase-7 activity was measured by using the CaspaseGlo kit from Promega.

The human NSCLC cell lines A549 and NCI-H1975 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 media containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO₂.
The bacterial strains and plasmids were obtained from the School of Life Science of Xiamen University, China. C086 (purity≥99%) was designed and synthesized by our laboratory (Figure 1B). C086 was dissolved in DMSO as a stock solution and diluted in culture media. Gefitinib was purchased from LC Laboratories (Woburn, MA, USA). Anti-Hsp90, anti-β-actin, anti-EGFR, anti-Ras, anti-C-Raf, anti-Akt, anti-P-Akt, anti-Mek, anti-P-Mek, anti-Erk½, anti-P-Erk, anti-C-Myc anti-Bax, anti-Bcl-2 (an apoptosis suppression protein), caspase-8, cleaved caspase-8, and the Apoptosis Antibody Sampler Kit containing PARP, cleaved PARP, caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7 were purchased from Cell Signaling Technology, Inc (Danvers, MA, USA). Annexin-V-APC/PI Apoptosis Detection Kit was purchased from Nanjing Keygen Biotech Co. Ltd (Nanjing, China). Propidium iodide (PI) was obtained from Sigma Aldrich.