HDACi facilitated DHA-induced apoptosis. A.

<p>Combination treatment with HDACi and DHA increased cell death. Cells were treated with 4 mM NaB, 1.25 μM SAHA, 10 μM DHA or combination of NaB/SAHA and DHA for 24 h. Cell viabilities were measured by MTT assay. <b>B.</b> The inhibitory effect of HDACi and DHA in combination on liver cancer cell growth was further confirmed by colony formation assay. One hundred of cells were seeded into 6-well plates for 7 d, and then cultured with either HDACi or DHA for another 7 d. Colonies were stained with 0.05% crystal violet. The number of colony in each well was counted and statistical analysis was performed. Data are presented as mean ± SD of three independent experiments. <b>C.</b> The effect of HDACi on DHA-induced apoptosis was measured by TUNEL assay, using <i>in situ</i> cell death detection kit. Hep G2 cells treated as described in <b>A</b> were subjected to TUNEL assay. Apoptotic cells were observed under fluorescent microscope. <b>D.</b> The effect of HDACi and DHA in combination was further confirmed by TUNEL assay, using flow cytometry. Percentage of apoptotic cells was calculated. <b>E.</b> Caspase 3 activation was involved in HDACi-mediated apoptosis in cells treated with DHA. The activity of caspase 3 in cells treated as described in <b>A</b> was determined and the relevant change was shown. For <b>A</b>, <b>B</b>, <b>D</b> and <b>E</b>, *<i>P</i><0.05, **<i>P</i><0.01, versus the DHA group.