Fig. 4

KRA-533 induces KRAS activation leading to apoptotic and autophagic cell death in human lung cancer cells. (a) and (b), A549, H157, Calu-1 and H292 cells were treated with increasing concentrations (0, 5, 10, 15 μM) of KRA-533 for 48 h. “0” means DMSO vehicle control. KRAS-GTP (active form of KRAS) was pulled down by Raf-1-RBD, followed by Western blot using KRAS antibody. Expression levels of KRAS, pERK, active caspase 3, PARP cleavage, Beclin1, LC3-I and LC3-II in total lysate were analyzed by Western blot. Apoptosis was determined by analyzing Annexin-V/PI binding by FACS. Data represent mean ± SD, *P < 0.05, **P < 0.01, by 2-tailed t test. (c) GFP-LC3 plasmids were transfected into A549, H157, Calu-1 and H292 cells. After 24 h, cells were treated with KRA-533 for 48 h. The percentages of LC3-GFP-transfected cells bearing LC3-GFP aggregates (LC3-FGPvac) were quantified as shown. Data represent mean ± SD, **P < 0.01, by 2-tailed t test