Fig. 2.

Cell cycle and cell death analyses in naïve and BETi-resistant MDA-MB-231 models. a. MDA-MB-231 and MDA-MB-231R cells were treated with JQ1, MZ1 and ARV-825 (0.4 μM) for 12 h. Then, cell cycle was evaluated by flow cytometry. Bar graphs show the percentage of cells in G0/G1, S, or G2/M cell cycle phases.b. MDA-MB-231 and MDA-MB-231R were treated with JQ1, MZ1, and ARV-825 for 12 h (0.4 μM). Then, cells were lysed and 50 μg of protein extract were analyzed by Western-blot with antibodies against proteins involved in cell cycle progression. Calnexin was used as loading control. c. Cell death produced by JQ1, MZ1 and ARV-825 (0.4 μM) in both cell lines was evaluated by flow cytometry with PI and Annexin V (AV) staining. Cells were classified in viable (AV -, PI -), early apoptotic (AV +, PI -), late apoptotic (AV +, PI +) and necrotic cells (AV -, PI+). d. MDA-MB-231 naïve and JQ1-resistant were pretreated with the pan-caspase inhibitor QVD (10 μM) for 45 min before being exposed to the drugs for 48 h. Cell death was analyzed by flow cytometry as described in C. e. Caspase 3 activity was measured by fluorescence (400/505 nm) and data were represented referred to control. *p < 0.05; **p < 0.01; ***p < 0.001. f. Cells were treated with JQ1, MZ1, and ARV-825 for 96 h (0.4 μM). Cells were then lysed and 50 μg of protein extract were analyzed by Western-blot with antibodies against proteins involved in apoptotic cell death. Calnexin was used as loading control