Figure 3

Gemcitabine causes translation repression in pancreatic cancer cells. (a) PANC-1 cells were incubated for 12 h in the absence or presence of 10 μM Gem or 0.5 μM ISRIB. Cell lysates were subjected to sucrose gradient centrifugation, and gradients were fractionated in-line with 254 nm UV absorbance measurement. Top fractions containing free ribosomal subunits are labeled as 40/43S, 60S. Mono ribosomes are labeled as 80S. Ribosomes bound to RNA fractions were labeled as polysomes. (b) PANC-1 cells were transfected with an empty vector or the GADD34 plasmid DNA, and incubated 24 h later with 10 μM Gem for 12 h. Cell lysates were analyzed for polysome profiles as in (a). (c) PANC-1 cells were incubated in the absence or presence of Gem or ISRIB+Gem and subjected to sucrose gradient centrifugation as in (a). Fractions were collected and luciferase (10 ng/ml) spiked into each fraction. RNA Isolated and ATF4 transcript levels were quantitated using qRT-PCR, and normalized to spike-in luciferase control. The percent total ATF4 transcript for each fraction is represented. Fractions 5, 6, and 7 corresponds to fractions with polysomes rich in translation. (d) PANC-1 cells were transfected with the TK-ATF4-Luc plasmid construct that with illustrated features: ATF4 5′UTR harboring uORF1 and uORF2, TK promoter and Luciferase coding region. At 24 h post transfection cells were incubated for 12 h in the absence or presence of 10 μM Gem and 0.5 μM ISRIB. Firefly luciferase units were measured and normalized to internal control Renilla luciferase activity. Data are the means ±S.D. of three experiments. *P<0.05, **P<0.005 compared with control, ^##P<0.001 compared with Gem+ISRIB. Panels a and b show representative data from three independent experiments