14-3-3ζ Is required for transcriptional activation of the HDAC1 gene by anisomycin and TSA. (A) Isoform-specific depletion of 14-3-3 proteins by RNA interference. HeLa cells were transfected with siRNAs against 14-3-3ɛ or ζ and either left untreated or stimulated with anisomycin and TSA (A/T). Whole-cell lysates were prepared and analyzed for 14-3-3 protein levels by immunoblotting. Equal loading was confirmed with a β-actin antibody. (B) 14-3-3 Depletion has no impact on the phosphoacetylation status of histone H3. HeLa cells, depleted of 14-3-3ɛ or ζ isoforms, and control cells were stimulated with TSA and anisomycin (sAn) for 1 h. ChIP assays were performed with the indicated antibodies. (C) Depletion of 14-3-3ζ interferes with transcriptional activation of the HDAC1 gene. HeLa cells were transfected with siRNAs as described for panel A. HDAC1 expression levels were determined by real-time RT–PCR, normalized to GAPDH levels and are depicted as fold increase compared with untreated samples, which were transfected with control siRNA. Values represent three independent experiments (mean±s.d.). Depletion of 14-3-3ζ interferes with the induction of HDAC1 by anisomycin and TSA treatment (lane 6, **P=0.002 and lane 8, *P=0.01, t-test), whereas 14-3-3ɛ depletion caused a more moderate reduction (lane 4, P=0.07, t-test).