Participation of mTORC2 in the p38 signaling pathway to regulate c-Myc expression after Cd exposure.

<p>HepG2 cells were treated with mTOR siRNA to reduce total mTOR expression then cultured in the presence or absence of 5 μM Cd for 4 h. Cells were harvested for Western blotting (A) and real-time PCR analysis (B). (C) Cells were treated with 100 nM rapamycin 1 h prior to the addition of 5 μM Cd then cultured for additional 4 h. Cellular proteins were extracted for Western blotting. (D) Cells were transfected with 50 nM control or Rictor siRNA. After treating with 5 μM Cd for 4 h, cells were harvested. Cell extracts were prepared for Western blotting. (E) c-Myc mRNAs from samples prepared as in (D) were quantified by real-time PCR. (F) Cells were treated with 50 μM p38 (SB202190) or JNK (SP600125) inhibitor for 1 h followed by the addition of 5 μM Cd. Cells were cultured for an additional 4 h. Cell extracts were prepared for Western blotting. Phospho-Akt was detected by the antibodies specific for the modification at Ser473. Actin was used as a loading control for immunoblotting. Phosphate proteins are designated with ‘p-‘ in front of the protein name. For RNA analysis, each value represents a mean ± standard deviation of three samples. Asterisks (*) indicate significant differences (<i>p</i> < 0.05) between the paired samples. Numbers underneath the Western blotting represent the amount of c-Myc relative to that of the loading control (actin).