Apicidin and docetaxel combined treatment stimulates proapoptotic signals in metastatic breast cancer cells. (A) FACS analysis of apoptosis (Annexin V+PI- and Annexin V+PI+) and necrosis (Annexin V-PI+) in MDA-MB-435, MDA-MB-231, and MCF-7 cell lines treated with medium or high doses of A, D, or A + D for 24 hours. All data are expressed as mean ± SD of five independent experiments. Statistical significance was performed by comparison of each treated cell group with Ctr cells, *P < .05. (B) Bcl-2 and Bax protein detection by Western blot analysis of lysates from 48-hour treated cells with medium and high doses of A and D, alone or in combination. Intensities of both Bcl-2 and Bax bands were measured and values normalized to actin are expressed as AU at the bottom of each panel; Bax/Bcl-2 ratio expression was evaluated on the normalized values and shown in the bottom panel. One representative experiment of three is shown. (C) Pro-caspase-8 and active caspase-8 protein expression of 48-hour treated cells with medium and high doses of A and D, alone or in combination. One experiment of two is shown.