Research Paper Volume 16, Issue 5 pp 4116—4137

Figure 7. Knockdown of PR55α does not result in senescence induction by IR in pancreatic cancer cells that lack p16 expression. (A) The CDKN2A locus, which encodes the p16 and p14 genes, is deleted from AsPC-1 pancreatic cancer cells [37]. Log-phase growing AsPC-1 cells were analyzed for the presence of p16 and p14 proteins with CD18/HPAF pancreatic cancer cells as a positive control. (B) AsPC-1 cells stably transduced with the Dox-inducible PR55α-shRNA or Control-shRNA were induced with 2 μg/ml Dox for 3 days to knock down PR55α. The cells were then exposed to 7 Gy IR, or left unirradiated (0 time point), and incubated for an additional 1, 4 and 7 days. The cells were analyzed by immunoblotting for the levels of PR55α, p53, and p21. GAPDH level was measured as an internal loading control. (C) The irradiated cells incubated for 7 days were analyzed for senescence by SA-β-gal activity assay and photographed. Scale bar = 1 μm.