Figure 5

Inhibition of ISR pathway completely reduces gemcitabine-induced Nupr1 increase in PANC-1 cells. (a) Nupr1 expression data from RNA-seq analysis and qRT-PCR is represented. **P<0.001 compared with control, ^##P<0.001 compared with ISRIB, ^$P<0.001 compared with Gem+ISRIB. PANC-1 cells were incubated with 0.5 μM ISRIB, 10 μM Gem, or ISRIB+Gem for 36 h, and RNA was analyzed for Nupr1 mRNA levels using qRT-PCR. Data are the means ±S.D. of three experiments. **P<0.001 compared with control, ^##P<0.001 compared with ISRIB, ^$P<0.001 compared with Gem+ISRIB. (b) Heat map for fold changes in gene expression in response to indicated drugs over control for genes involved in Nupr1 network, based on Nupr1 network genes collected from Ingenuity Pathway Analysis as described in Materials and Methods. (c) PANC-1 cells stably expressing either control-shRNA or PERK-shRNA and PANC-1 cells with ATF4 siRNA were incubated with 10 μM Gem for 24 h, and RNA was analyzed for Nupr1 mRNA expression levels. Data are the means ±S.D. of three experiments. **P<0.001 compared with control-shRNA and PERK-shRNA, ^##P<0.001 compared with PERK-shRNA, *P<0.001 compared with control-shRNA, ^$P<0.001 compared with PERK-shRNA in the presence of Gem. (d) PANC-1 cells were transfected with indicated siRNA, and incubated 48 h later in the absence or presence of 10 μM Gem for 24 h. Caspase-3 activity was measured using Casp3/7 glow assay. Data are the means ±S.D. from three independent experiments. **P<0.001 compared with control, ^##P<0.001 compared with sham-siRNA control and sham-siRNA with Gem, ***P<0.001 compared with sham-siRNA control, ATF4 siRNA control, and sham-siRNA with Gem, ^$P<0.001 compared with sham-siRNA control, Nupr1-siRNA control, and sham-siRNA with Gem. (e) Antiapoptotic factor BEX2 and (g) BCL2A1 expression data from RNA-seq analysis and qRT-PCR analysis are shown; **P<0.001 compared with control and ISRIB, ^##P<0.001 compared with Gem, ^$P<0.001 compared with Gem+ISRIB. (f) PANC-1 cells were incubated with 0.5 μM ISRIB or 10 μM Gem for 36 h and protein lysates were analyzed by immunoblotting for Nupr1 and BEX2 using specific antibody. ERK2 served as loading control. Nupr1 protein levels were quantified from the blots and fold changes in expression are shown. Data are the means±S.D. of three experiments. *P<0.01 compared with control, ISRIB, and Gem+ISRIB. (h and i) PANC-1 cells expressing ATF4 siRNA were incubated with 10 μM Gem for 24 h, and RNA was analyzed for (h) BEX2 mRNA and (i) BCL2A1 expression levels. Data are the means ±S.D. of three experiments. **P<0.001 compared with control siRNA and ATF4 siRNA, ^##P<0.001 compared with ATF4 siRNA, *P<0.001 compared with control siRNA, ^$P<0.001 compared with ATF4 in the presence of Gem