Gain- and Loss-of-Function Studies of MDM2

Based on the expression of MDM2 in LAC cell lines, we selected PC9 cells for gain-of-function studies and the H1975 cells for loss-of-function studies. The lentivirus-packaged MDM2 overexpression vector, MDM2-shRNA, and all negative controls were constructed and verified as previously described.28 (link) These procedures were performed by GenePharma (Shanghai, China). Stably transfected cell lines were screened with 0.6 μg/mL puromycin (Sigma). Knockdown of Smad2 and Smad3 in H1975 cells was performed by transfecting cells with specific siRNAs (Santa Cruz, CA, USA). DNA sequences used in this study are given below. Scrambled negative siRNA sequence: 5′-TTC TCC GAA CGT GTC ACG UTT-3′; Smad2 siRNA: 5′-AUC UAA UCG UCC UGU UUU CUU TT-3′; Smad3: 5′-UCU UCU UGA GUU UCU UGA CCA TT-3′; MDM2 shRNA: 5′-UUG GUA UUG CAC AUU UGC CUG-3′. Lipofectamine 2000 reagents (Invitrogen) were used for cell transfection according to the manufacturer’s instructions.