miR-183 repression requires MYCN and HDAC2. (A) MYCN and HDAC2 expression in the IMR-32 cell line, harboring a tetracycline-inducible MYCN shRNA expression construct, is shown after 48 h of treatment with or without tetracycline (Tet). ß-actin served as a loading control. (B) miR-183 expression was analysed by qRT-PCR in the same neuroblastoma cell model after MYCN shRNA induction. Bars show mean miR-183 expression relative to solvent-treated cells ± SD. (C) ChIP analysis with a HDAC2-specific antibody revealed less HDAC2 recruitment (gray bar) to the miR-183 promoter region in lysates of cells treated as in (A) after MYCN depletion. Mean enrichment detected by miR-183 promoter qRT-PCR relative to solvent-treated cells (±SD) is shown. (D) ChIP analysis investigating MYCN recruitment to the miR-183 promoter region upon Panobinostat treatment. BE(2)-C cells were treated with Panobinostat or solvent for 2 h or 24 h. Bars represent mean MYCN recruitment to the miR-183 promoter region (±SD) relative to solvent-treated cells at each time point (=100%) measured by miR-183 promoter region qRT-PCR. Western blot for MYCN expression and histone H3 pan-acetylation with the H3 and ß-actin loading controls are shown below. *P ≤ 0.05; n.s., not significant.