Fig. 2. Vulnerability due to RB loss.

A Heat map representing the differential effect of drugs (250 nM) from cluster 2 on the viability of MCF7-WT and RB-del cells as determined by CTG assay. B Differential effects of alisertib and pemetrexed at the indicated concentrations on the RB-proficient and RB-deficient MCF7, T47D and CAMA-1 cells following 6 days of exposure. Bars represent mean and SD from triplicates. Experiments were performed at 2 independent times (**p < 0.01, ***p < 0.001 as determined by 2-way ANOVA). C Western blot analysis to demonstrate the effect of pemetrexed (Pem) (200 nM) and gemcitabine (Gem) (100 nM) on PARP cleavage in RB-proficient and RB-deficient MCF7 and T47D cells after 72 h treatment. Effect of alisertib at two different concentrations on cleaved PARP in MCF7, T47D and CAMA-1 WT and RB-del cells following 72 h treatment. D Heat maps to demonstrate the synergistic effect on the inhibition of cell viability by different pairwise combination of the indicated drugs in MCF-WT and MCF7-RB-del cells following 6 days of exposure. E CTG assay on MCF7, T47D and CAMA-1 WT/RB-del cell lines following the treatment with the indicated drugs at indicated concentrations as single agents and combination for up to 6 days. Mean and SD were calculated from triplicates (**p < 0.01, ***p < 0.001 as determined by 2-way ANOVA). F Western blot analysis on MCF7-WT, MCF7-RB-del, T47D-WT and T47D-RB-del cells that were reverse transfected with RNAis to silence WEE1 and AURKA. Viability of MCF7-WT and RB-del cells were determined by CTG assay following the knockdowns of WEE1 and AURKA up to 6 days. Bars represent mean and SD from triplicates. Experiments were performed at two independent times (**p < 0.01 as determined by two-way ANOVA). Western blots represent replicates from two independent experiments.