B7-H6 expression is reduced by inhibitors of class I HDACs and by HDAC2 or HDAC3 knockdown. HeLa cells were treated for 16 hours with the following HDACi: pan-HDACi SAHA and TSA, class I–specific VPA, and apicidin as well as HDAC8-specific Cpd2. DMSO was used as a solvent control for all inhibitors except for VPA, which was dissolved in water. (A) HeLa cells were stained with anti-B7-H6 mAb 1.18 or isotype control for flow cytometry. Dead cells were excluded by gating on 7-AAD⁻ cells. The mean geometric mean ± standard deviation (SD) of at least 3 independent experiments is shown. (B) B7-H6 mRNA expression in treated HeLa cells was determined by qRT-PCR. The mean (% expression of B7-H6 relative to GAPDH) ± SD is shown for at least 2 independent experiments. The values from medium (control for VPA) and DMSO (control for the other inhibitors) were set as 100%, respectively. (C) HeLa cells were transfected with 2 different siRNAs (1 and 2) targeting HDAC1, HDAC2, or HDAC3 and a negative control (NC) siRNA. Knockdown was confirmed by western blot with β-actin staining as a loading control. Cropped blot images are shown. For flow cytometry, cells were stained with anti-B7-H6 mAb 1.18 or isotype control as in A. Representative histograms and the mean % B7-H6 surface expression compared with NC siRNA (set as 100%) ± SD of at least 2 experiments are shown. (D) B7-H6 mRNA expression in siRNA-transfected HeLa cells was determined by qRT-PCR. Mean (% expression of B7-H6 relative to GAPDH) compared with the NC siRNA (set as 100%) ± SD is shown from at least 2 experiments. ns, not significant; *P ≤ .05; **P ≤ .01; and ***P ≤ .001.