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. 2018 Aug 3;9(60):31572–31589. doi: 10.18632/oncotarget.25803

Figure 4. PD0325901 down-regulation of CDK2 activation in palbociclib-resistant cells is dependent on p27Kip1.

Figure 4

A. Kinase activity of CDK2 (left panel), CDK4 (middle panel) and CDK6 (right panel) in H358-PR250 cells treated with vehicle (DMSO) or 100 nM PD0325901 in the absence or presence of RB protein substrate. B. Western blot analysis assessing RB phosphorylation and the expression of p21Waf1/Cip1 and p27Kip1 in parental H358 and H358-PR250 cells treated with vehicle (-) or 100 nM PD0325901 (+) for 24 hours. C. p27Kip1 mRNA expression assessed by RT-PCR in parental H358 and H358-PR250 cells treated with vehicle or 100 nM PD0325901 without or with siRNA-mediated depletion of p27Kip1. Drug treatments were for 24 hours. D. Co-immunoprecipitation of p27Kip1 with CDK2, CDK4 or CDK6 in H358-PR250 cells treated with vehicle or 100 nM PD0325901 for 24 hours. E. Coimmunoprecipitation of p27Kip1 with CDK7 in parental H358 and H358-PR250 cells treated with vehicle or 100 nM PD0325901 for 24 hours. CDK7-dependent phosphorylation of CDK2 was also assessed using a phospho-specific antibody to phospho-CDK2 [T160]. Significance was assessed using a two-way ANOVA with Bonferroni post-analysis with ** P < 0.01, *** P < 0.001; Results represent the average of 3 independent experiments.