Figure 1
Gemcitabine induces the integrated stress pathway in pancreatic cancer cells. (a) AsPC-1 and PANC-1 cells were incubated with 0.5 μM ISRIB or 10 μM Gem for indicated times and protein lysates were analyzed by immunoblotting for phospho-eIF2, total eIF2, and ATF4 using specific antibodies. ERK2 served as loading control. (b) Empty vector or GADD34 expression plasmid DNA was transfected into PANC-1 cells, which were incubated 24 h later with 10 μM Gem. Protein lysates were analyzed by immunoblotting. (c) PANC-1 cells were incubated for 36 h with 0.5 μM ISRIB or 10 μM Gem and protein lysates were analyzed by immunoblotting for ATF4, ATF3, GADD34, and CHOP using specific antibodies. ERK2 served as loading control. (d) PANC-1 cells were incubated with 0.5 μM ISRIB or 10 μM Gem for 24 h. ATF4, CHOP, ATF3, and GADD34 mRNA levels were measured using qRT-PCR. Relative actin mRNA levels were used for normalization. Data are the means±S.D. from three experiments. *P<0.05, **P<0.001 compared with control, ##P<0.01 compared with ISRIB, and $P<0.05 compared with Gem+ISRIB. (e) MEFs were incubated for 6 h with 10 μM Gem in the absence and presence of ISRIB. As a positive control, MEFs were also incubated for 6 h with thapsigargin (0.1 μM). (f) Wild-type MEFs or MEFs mutated for eIF2α at serine 51 (to alanine) were incubated with 10 μM Gem for indicated time point and analyzed by immnuoblotting. Panels (a, b, d and e) show representative data from three independent experiments
