Combination treatment of ACY-241 and JQ1 synergistically decreases HNSCC cell viability. (A,B) ACY-241 and JQ1 were treated alone or in combination at a 2:1 ratio to 2A3 cells and FaDu cells. Cell viability was measured by CCK-8 assay (48 h). Synergism of ACY-241 and JQ1 was determined by combination index (CI) using Chou–Talalay method. (C,D) Inhibitory enzymatic effects of ACY-241 and JQ1 were confirmed by immunoblot analysis of acetyl α-tubulin and c-Myc, respectively. 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. Values represent mean ± SD (n = 3). * p < 0.05, ** p < 0.01, or *** p < 0.001 vs. DMSO control, $
p < 0.05, $$
p < 0.01, or $$$
p < 0.001 vs. ACY-241-treated group, #
p < 0.05, ##
p < 0.01, or ###
p < 0.001 vs. JQ1-treated group. ns = not significant.