Downregulation of B7-H6 by HDACi is associated with reduced B7-H6 reporter activity and decreased histone acetylation. (A) HeLa cells were transfected with a luciferase (GLuc) reporter construct containing the B7-H6 5′-UTR (−1270 to +208, see insert) or a VC followed by DMSO/SAHA treatment. The mean (fold change luciferase activity relative to VC) ± standard deviation (SD) is shown for 1 representative experiment out of 2. The value obtained in the VC sample was set as 1. (B) ChIP assays were performed using chromatin from HeLa cells for IP with the indicated mAbs or isotype control. Binding of HDAC1-3 to a region including the transcription start site in the B7-H6 5′-UTR (see insert in B) was assessed. The mean (signal relative to the input) ± SD of duplicates from 1 experiment out of 4 is shown. (C) Acetylated histone H3 and H4 were analyzed by ChIP 16 hours after DMSO or SAHA treatment. The mean (signal relative to the input) ± SD of duplicates from 1 experiment out of 3 is shown. (D) The binding of HDAC1-3 and the HAT CBP was analyzed by ChIP after DMSO/SAHA treatment. The mean (signal relative to the input) was calculated as: (signal with indicated mAb) – (signal with isotype control); ± SD from at least 3 experiments is shown. (E) B7-H6 mRNA levels were determined after DMSO/SAHA treatment in the presence of actinomycin D (ActD). The mean (% expression of B7-H6 relative to GAPDH) ± SD is shown for each time point after ActD addition of 2 experiments. The value before ActD treatment was set as 100%. ns, not significant; *P ≤ .05; ***P ≤ .001.