Journal: Oncogene

Article Title: RB loss determines selective resistance and novel vulnerabilities in ER-positive breast cancer models

doi: 10.1038/s41388-022-02362-2

Figure Lengend Snippet: 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.

Article Snippet: Palbociclib, Alisertib, and Birinapant were purchased from MedChemExpress (NJ, USA).

Techniques: Drug discovery, Western Blot, Live Cell Imaging, BrdU Incorporation Assay, Mutagenesis

Journal: Oncogene

Article Title: RB loss determines selective resistance and novel vulnerabilities in ER-positive breast cancer models

doi: 10.1038/s41388-022-02362-2

Figure Lengend Snippet: A Heat maps representing the relative cell viability of MCF7-WT, MCF7-RB-del, T47D-WT and T47D-RB-del cells in the presence of drug library (100 nM) following the pretreatment with birinapant (250 nM) from clusters 1 & 2. B Cell viability assays from MCF7 and T47D WT/RB-del that were treated with birinapant in combination with the indicated drugs at the indicated concentrations up to 6 days. Mean and SD were calculated from triplicates. Experiment was done at 2 independent times. (*p < 0.05, **p < 0.01 as determined by two-way ANOVA). C Crystal violet staining from MCF7-WT, MCF7-RB-del, T47D-WT and T47D-RB-del cells that were treated with Alpelisib (100 nM) in combination with DMSO or birinapant (500 nM). Cells were allowed to form colonies up to 15 days. D Live cell imaging to monitor the proliferation of MCF7-WT and RB-del cells that were treated with DMSO, birinapant (500 nM) and palbociclib (50 nM) + fulvestrant (100 nM) ± birinapant. The live cell imaging was terminated, and the cells were subjected to CTG assay to determine the viable cells. Mean and SD were calculated from 6 replicates. (**p < 0.01, ***p < 0.001 as determined by two-way ANOVA).

Article Snippet: Palbociclib, Alisertib, and Birinapant were purchased from MedChemExpress (NJ, USA).

Techniques: Drug discovery, Staining, Live Cell Imaging, CTG Assay