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. Author manuscript; available in PMC: 2023 Nov 27.
Published in final edited form as: Oncogene. 2022 Jun 9;41(27):3524–3538. doi: 10.1038/s41388-022-02362-2

Fig. 1. RB-mediated cell cycle arrest by standard care of therapies.

Fig. 1

A Heat map representing the relative growth rate of MCF7 and T47D cells in the presence of drug library at two different concentrations (100 nM and 500 nM) from cluster 1. B Western blot analysis on the indicated proteins from MCF7 and T47D cells that were treated with everolimus (250 nM) and alpelisib (250 nM) up to 48 H. C Western blot analysis to investigate the effect of alpelisib (250 nM) on cell cycle proteins from T47D and T47D-D1/K4 that were treated up to 48 H. D Live cell imaging to monitor the growth of MCF7-WT, MCF7-D1/K4, T47D-WT, and T47D-D1/K4 cells treated with different pairwise combination of palbociclib (50 nM), fulvestrant (100 nM), everolimus (125 nM) and alpelisib (125 nM). Error bars represent mean and SD from triplicates. Experiments were done at two independent times. E Western blot on the indicated proteins from MCF7-WT, MCF7-D1/K4, T47D-WT and T47D-D1/K4 that were treated with palbociclib (50 nM) + fuvlestrant (100 nM) and alpelisib (125 nM) + fulvestrant (100 nM) for 48 h. F Growth curves illustrating the effect of indicated combination treatments involving palbociclib (50 nM), fulvestrant (100 nM), everolimus (125 nM) and alpelisib (125 nM) in MCF7-RB-del and T47D-RB-del cells. Mean and SD were calculated from triplicates. G Western blot analysis to demonstrate the differential effect of palbociclib (50 nM) + Everolimus (125 nM) and palbociclib (50 nM)+ alpelisib (125 nM) on cell cycle proteins. H Heat map to demonstrate the synergistic effect between the combination of palbociclib with everolimus (Palb/Evero) and alpelisib (Palb/Alpe) on BrdU incorporation at the indicated doses in MCF-WT, MCF7-RB-del, T47D-WT, and T47D-RB-del cells. The values in heat map represent the mean from triplicates. BrdU incorporation was determined in CAMA-1 WT and CAMA-1-RB-del cells treated with the palbociclib (palbo) in combination with everolimus (evero) and fulvestrant (fulve) at indicated concentrations for 72 h. Error bars represent SD from triplicates (*p < 0.05, **p < 0.01 as determined by two-way ANOVA). I PFS of patients receiving CDK4/6i in combination with endocrine therapy with tumors harboring WT RB (n = 68) and RB-deletion mutation (n = 3). All p values were indicated.