Combination treatment of ACY-241 and JQ1 synergistically induces apoptosis in HNSCC. (A,B) Immunoblot analysis of pro-apoptotic proteins (PARP, Cas-3) and anti-apoptotic proteins (XIAP, Bcl-xL) in 2A3 and FaDu cells. α-tubulin and GAPDH were used as loading controls. Protein levels were quantified relative to the loading control. Total protein was extracted after 24 h of ACY-241 (4 μM) or JQ1 (2 μM) treatment alone or in combination. (C,D) Flow cytometry analysis of 2A3 and FaDu cells. Cells were treated with 0.2% DMSO, ACY-241 (4 μM), or JQ1 (2 μM) alone or in combination for 72 h. 2A3 and FaDu cells were stained with annexin V and PI for 15 min. Values represent mean ± SD (n = 3). * p < 0.05, ** p < 0.01, or *** p < 0.001 vs. DMSO control, $
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p < 0.001 vs. ACY-241-treated group, #
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p < 0.001 vs. JQ1-treated group.