Systematic shRNA-mediated screen of individual Hdac isoforms uncovers sensitivity of Eµ-Myc lymphoma cells to depletion of Hdac3. Eµ-Myc lymphoma cells (no. 4242) were transduced with constitutive (pLMS) or dox-inducible (pTRMPV-Neo) retroviral vectors expressing shRNAs targeting HDACs1, 2, 3, or 6. Cells were isolated by FACS and then serially passaged (±dox) for up to 15 days. The percentage of shRNA-expressing cells (GFP⁺, pLMS; or Venus⁺/dsRed⁺, pTRMPV-Neo) were assessed by flow cytometry (days 1, 3, 5, 7, 9, 11, 13) and data were normalized to day 1, as depicted. (A) Constitutive depletion of individual Hdacs in Eµ-Myc cells in vitro (n = 2 biological replicates). Data are presented as mean ± SEM. (B) qRT-PCR was used to determine the efficiency of Hdac depletion in Eµ-Myc cells (day 3). Results were normalized to L32, whereas relative mRNA level in control cells (shScr) was set to 1 (n = 3-4 biological replicates). Data are presented as mean ± SEM. (C) Schematic representation of the dox-inducible vector system (pTRMPV-Neo). Representative dot plots demonstrating: (i) nontransduced (Venus⁻) cells; (ii) transduced (Venus⁺) cells; and (iii) shScr-expressing (Venus⁺/dsRed⁺) Eµ-Myc cells from at least 3 independent experiments. (D) Dox-inducible depletion of individual Hdacs in Eµ-Myc tumor cells in vitro (no. 4242; n = 2 independent experiments; days 1, 3, 5, 7, 9, 11, 13, 15). Data are presented as mean ± SEM. (E) Western blotting was used to demonstrate the efficiency of inducible Hdac depletion in Eµ-Myc cells (day 3). Hyperacetylated tubulin (Ac tubulin) was used as a surrogate readout for Hdac6 depletion, whereas changes to its levels in non-shHdac6 samples represent background variability (for example shHdac1). A representative experiment from 3 biological replicates is shown. Molecular weights of individual proteins are to the left of each blot.