RNAi-mediated screen of all 11 classical Hdac isoforms in AML cells (MLL-AF9;Nras^G12D) demonstrates a unique dependency on Hdac3 expression. Eleven Hdac isoforms were depleted in MLL-AF9;Nras^G12D AML using multiple shRNAs per Hdac. Transduced AML cells (GFP⁺, shRNA-expressing) were mixed with nontransduced cells and followed for 10 days for cell representation by flow cytometry. (A) Relative depletion of GFP⁺ AML cells following constitutive (pLMN) depletion of individual Hdacs during 10 days of serial culture (Hdac3 shown in red). Data are plotted as fold change (GFP% day 2/GFP% day 12). A single experimental screen was undertaken with 4-5 distinct shRNAs per gene represented by individual bars. Dotted line depicts a 3-fold depletion cut off. (B) The efficiency of Hdac3 knockdown in AML cells was validated by qRT-PCR (day 2). Results were normalized to glyceraldehyde-3-phosphate dehydrogenase, whereas relative messenger RNA level in control cells (shRen.713) was set to 1 (n = 3-4 independent biological replicates). Data are presented as mean ± SEM. (C) Dox-inducible depletion of Hdac3 in AML. Each series of bars demonstrates a time course: days 1, 2, 4, 6, 8, 10, 12, and 14. The percentage of shRNA- expressing cells (Venus⁺/dsRed⁺) was normalized to day 1. Data are representative of a single experiment using 3 individual shRNAs to Hdac3 (shHdac3.987, shHdac3.1491, shHdac3.1037). Established control shRNAs to Myc, Myb, and MLL/AF9 cofactor Men1 were included for comparison.