FIG. 3.

Signaling changes by OSI-027 in pancreatic cancer cells. Human pancreatic cancer cell lines (PANC-1 or MIA PaCa-2) were either left untreated (“C”), or treated with OSI-027 (50 nM) for an indicated time (6/24 h), expression of listed proteins was tested by western blots (A, B, and E). PANC-1 (C) or MIA PaCa-2 (D) cells were treated with OSI-027 (50 nM), with or without MEK/ERK inhibitor PD98059 (500 nM), U0126 (500 nM), or MEK162 (100 nM) for 72 h, cell survival was tested by CellTiter-Glo assay. PANC-1 or primary pancreatic cancer cells were preadded with 3-methyladenine (3-MA, 0.5 mM), chloroquine (Cq, 10 μM), or ammonium chloride (NH₄Cl, 10 mM) for 1 h, followed by OSI-027 (50 nM) treatment, cell survival was tested by CellTiter-Glo assay 72 h after OSI-027 treatment (F, G). PANC-1 cells, transfected with scramble control siRNA (“sc-RNAi”) or Beclin-1 siRNA (100 nM each, 36 h), were either left untreated (“C”), or treated with OSI-027 (50 nM) for 72 h, expression of Beclin-1 and GAPDH was tested by western blots (H), cell survival was also tested (I). Phosphorylation or expression of indicated proteins were quantified, and was normalized to the nonphosphorylated kinase or the loading control (GAPDH). LC3-II intensity was normalized to that of LC3-I to calculate the LC3-I/II conversion. For (C, D, F, G, and I): Data are expressed as mean±SE, experiments were repeated five times. n=5 for each assay.*p<0.05 versus “C” group. ^#p<0.05 versus OSI-027 only group (C, D, F, G). ^#p<0.05 versus “sc-RNAi” group (I).