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Fig. 6 | Journal of Experimental & Clinical Cancer Research

Fig. 6

From: PES1 promotes BET inhibitors resistance and cells proliferation through increasing c-Myc expression in pancreatic cancer

Fig. 6

CDK5 inhibitors destabilize PES1 and increase the sensitivity of pancreatic cancer cells to BET inhibitors. a and b, PANC-1 and BxPC-3 cells were treated with DMSO, Palbociclib (5 uM), MK2206 (5 uM), MK1775 (0.5 uM), Roscovitine (1 uM), SB203580 (0.5 uM), RAD001 (5 nM), Dinaciclib (5 nM), PD0325901 (1 nM), JQ1 (1 uM) for 48 h. Cells were harvested for Western Blotting analysis (a) and RT-qPCR analysis (b). Data presented as Means ± SD (n = 3). N.s., not significant. c and d, PANC-1 and BxPC-3 cells treated with different dose of Dinaciclib for 48 h. Cells were harvested for Western Blotting analysis (c) and RT-qPCR analysis (d). Data presented as Means ± SD (n = 3). N.s., not significant. e, schematic diagram depicting that the CDK5 phosphorylation consensus motif of PES1. f, the whole cell lysates of PANC-1 were harvested for Western Blotting analysis. g and h, pancreatic cancer cell lines (PANC-1 and BxPC-3) were infected with indicated construct. After 72 h, were harvested for Western Blotting analysis and Rt-qPCR analysis. Data presented as Means ± SD (n = 3). N.s., not significant. i, PANC-1 cells were transfected with indicated plasmids. After 24 h, cells were treated with Cycloheximide (CHX) and cells were collected for Western Blotting analysis at different time points. j, PANC-1 cells were transfected with indicated plasmids. After 72 h, cells were harvested for Western Blotting analysis. k, Wild-type Flag-PES1 and mutant Flag-PES1 (S424A) proteins were translated in vitro. These proteins were purified with Pierce Protein G Agarose and primary antibody (Flag-tag antibody) in the cold room overnight. Then, the purified proteins were incubated with activated CDK5/p25 and ATP-γ-S. Thiophosphate ester was detected by Western Blotting analysis. l, PANC-1 cells were transfected with indicated plasmids. After 24 h, cells were treated with Cycloheximide (CHX) and cells were collected for Western Blotting analysis at different time points. m, PANC-1 cells were transfected with indicated plasmids. After 24 h, cells were harvested for Western Blotting analysis. n, PANC-1 cells were infected with indicated shRNA. After 72 h, cells were harvested for Western Blotting analysis and RT-qPCR analysis. Data presented as Means ± SD (n = 3). N.s., not significant; **, P < 0.01; ***, P < 0.001. o, PANC-1 cells were infected with indicated shRNA. After 72 h, cells were harvested for Western Blotting analysis and RT-qPCR analysis. Data presented as Means ± SD (n = 3). N.s., not significant; ***, P < 0.001. p-r, PANC-1 cells were treated with indicated drugs. Cells were collected for colony formation assay (p) and xenografts assay (q and r). Data presented as Means ± SD (n = 3 for O, n = 5 for p and q). ***, P < 0.001

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