Fig. 5: Cellular response to INX-315. | Nature Communications

Fig. 5: Cellular response to INX-315.

From: Discrete vulnerability to pharmacological CDK2 inhibition is governed by heterogeneity of the cancer cell cycle

Fig. 5

A Effect of INX-315 on the proliferation of indicated cell lines. Error bars indicate mean and SD from triplicates. B Bivariate flowcytometry analysis in MCF7 and MiaPaCa-2 cell lines following the treatment with different concentrations of INX-315 for 48 h. C Western blotting on the indicated proteins from MCF7, MiaPaCa-2 and 3226 cell lines following the treatment with INX-315 for 48 h. D Biochemical analysis to determine the differential effect of INX-315 from MB157-WT and MB157-RB-del cell lines. E Immunoprecipitation of CDK2 from MCF7 and MIA PaCa-2 cells following the treatment with INX-315 (500 nM) for 48 h. Coimmunoprecipitated proteins including cyclin E1, cyclin A and cyclin B1 from whole cell lysate (Input), CDK2-complex and flow-through were determined by western blotting. F Column graphs depicting the fraction of the indicated cyclins in complex with CDK2 and CDK1 from MCF7 and MiaPaCa-2 cell lines in the absence and presence of INX-315 (500 nM), which was determined based on densitometry analysis from the western blot in Fig. 5E. Error bars were determined based on mean and SEM. Statistical Analysis was determined based on two-tailed student t-test. In MCF7 the experimental replicates are as follows: for cyclin E1/CDK2: n = 3, cyclin A/CDK2: n = 4; cyclin B1/CDK2: n = 3; cyclin A/CDK1: n = 2, cyclin B1/CDK1: n = 3. The p value for cyclin E1/CDK2 complex is 0.0367 and the p value for cyclin A/CDK2 complex is 0.0082. For MiaPaCa-2 cells, the experimental replicates include, cyclin E1/CDK2: n = 3; cyclin A/CDK2: n = 6; cyclin B1/CDK2: n = 3; cyclin A/CDK1: n = 2; cyclin B/CDK1: n = 3. The p value for cyclin E1/CDK2 complex is 0.0435 and the p value for cyclin A/CDK2 complex is 0.0293. G In vitro kinase reaction to determine the catalytic activities of CDK2 and CDK1 in the presence of INX-315 at different concentrations. The kinase reactions were carried out in the presence of ATP (500 µM). The C-terminal peptide from RB was used as substrate for the kinases. The phosphorylation status of RB was determined by western blotting. H Densitometry analysis of pRB western blot from the Fig. 5G represents catalytic activity of CDK2 and CDK1 and plotted against the concentration of INX-315. Error bars represent mean and SEM from 3 independent biological replicates. Statistical analysis was performed based on 2-way Anova (*** represents p value < 0.0001). Western blotting was done at two independent times (C & D). Source data are provided as a source data file.

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