Fig. 3.

EMS inhibits p53 expression at the translational level. (A) A549 cells were infected with lentiviruses expressing control shRNA, EMS shRNA#1, or EMS shRNA#2. (B) A549 cells were infected with lentiviruses expressing control (pSin) or EMS (pSin-EMS). pSin is a lentiviral expression vector. Forty-eight hours after infection, cell lysates and total RNA were analyzed by western blotting and real-time RT-PCR, respectively. Data shown are mean ± SD (n = 3). ns., no significance. *P < 0.05; **P < 0.01; ***P < 0.001. (C and D) (C) A549 cells were infected with lentiviruses expressing control shRNA or EMS shRNA. (D) A549 cells were infected with lentiviruses expressing control (pSin) or EMS (pSin-EMS). Forty-eight hours after infection, cells were treated with or without MG132 (20 μM) for 4 h. Cell lysates were then analyzed by western blotting. LE and SE indicate long-time exposure and short-time exposure, respectively. (E) A549 cells expressing control or EMS were treated with cycloheximide (CHX; 20 μg/mL) for the indicated periods of time, followed by western blot analysis. The band intensities were quantified using ImageJ software. The ratio of p53 to GAPDH is presented in SI Appendix, Fig. S1C. (F) A549 cells expressing control shRNA, EMS shRNA#1, or EMS shRNA#2 were treated with CHX (20 μg/mL) for the indicated periods of time, followed by western blot analysis. The band intensities were quantified using ImageJ software. The ratio of p53 to GAPDH is presented in SI Appendix, Fig. S1D. (G) A549 cells expressing control shRNA, EMS shRNA#1, or EMS shRNA#2 were pulse labeled with [³⁵S]cysteine/methionine for 90 min. Cell lysates were then immunoprecipitated with anti-p53 antibody and analyzed by SDS-PAGE, followed by autoradiography. (H) A549 cells expressing control or EMS were pulse labeled with [³⁵S]cysteine/methionine for 90 min. Cell lysates were then immunoprecipitated with anti-p53 antibody and analyzed by SDS-PAGE, followed by autoradiography. ctrl, control; IP, immunoprecipitation.