Fig. 2: Combined targeting of ER, CDK4/6 and AKT efficiently inhibits cyclin D/CDK4-6/Rb and PI3K/AKT-mTOR pathways in ER+ breast cancer cell lines.

Apoptotic levels were determined by evaluating the presence of cytoplasmic nucleosomes in MCF-7 (A), T47D (B), and ZR-75-1 (C) cells using an ELISA cell death detection assay. Data are shown with error bars representing mean ± SEM of three biological replicates. Asterisks indicate significant differences in the one-way ANOVA test (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, and ****p < 0.0001). D Western blot analysis of apoptosis markers in the three fulvestrant-resistant breast cancer models. Densitometry analysis of Western blot bands of cleaved-PARP was performed using ImageJ software. Data are shown as the area under the curve (AUC) normalized to loading control. E Western blot analysis of key signal transduction proteins in the three fulvestrant-resistant breast cancer models. F Western blotting analysis of p-AKT (S473) and total AKT expression in the three fulvestrant-resistant breast cancer models. Densitometry analysis of Western blot bands of p-AKT (S473) and total AKT was performed using ImageJ software. Data are shown as the area under the curve (AUC) normalized to loading control. GAPDH was used as a loading control for Western blotting analysis. For all Western blots, a representative of two biological replicates is shown. Both apoptosis assay and harvesting protein for Western blotting analysis were performed 3 days after treatment with fulvestrant (Fulv, 100 nM), CDK4/6 inhibitor (CDK4/6i, palbociclib, 200 nM in the MCF-7 and T47D cell models; 5 µM in the ZR-75-1 cell model) and AKT inhibitor (AKTi, capivasertib, 500 nM in the MCF-7 cell model; 200 nM in the T47D model; 150 nM in the ZR-75-1 cell model).