Depletion of Hdac3 in Eµ-Myc lymphoma induces an antiproliferative response that is not affected by genetic or pharmacological apoptosis inhibitors or by loss of p21^WAF1/CIP1. Eµ-Myc lymphoma cells were transduced with pTRMPV-Neo vectors expressing shHdac3 or control shScr cassettes, isolated by FACS (100% GFP⁺), serially passaged (±dox) for up to 15 days. (A) The induction of apoptosis was assessed by phosphatidylserine externalization (Annexin V⁺) using flow cytometry. As a positive control, we treated Eµ-Myc lymphoma cells with vorinostat (24 hours, 1 µM). Data are presented as percentage of Annexin V positive cells (mean ± SEM, n = 2 biological replicates). Individual bars demonstrate day of analysis (days 1, 3, 5, 7, 9, 11). (B-C) Eµ-Myc lymphoma cells (no. 107) were stably transduced with pMSCV.Bcl-2-mCherry to overexpress pro-survival Bcl-2. Eµ-Myc.Bcl-2 lymphoma cells were then transduced with constitutive vectors expressing shRNAs against Hdac3 or shScr, serially passaged, and assessed by (B) competitive proliferation assay where the percentage of GFP⁺ cells were normalized to day 1 and individual bars represent days of analysis (days 1, 3, 5, 7, 9, 11, 13; 3 individual shRNAs were tested; 2 biological replicates) and (C) HDAC3 depletion was confirmed by western blot. A representative experiment from 3 biological replicates is shown. Molecular weights of individual proteins are to the left of the blot. (D) Caspase activation was inhibited by treating Eµ-Myc lymphoma cells expressing dox-inducible shRNAs with pan-caspase inhibitor QVD (10 µM, n = 3 biological replicates); cell growth was assessed using competitive proliferation assays (±dox). Data are presented as mean percentage of shRNA-expressing cells (Venus⁺/dsRed⁺, dox-treated only, days 3, 5, 7, 9) and normalized to day 3 ± SEM. The activity of QVD was confirmed in Eµ-Myc cells treated with vorinostat (1 µM) ± QVD (presented as percentage viable cells). The proliferation of Eµ-Myc cells depleted of Hdac3 was measured using a carboxyfluorescein diacetate succinimidyl ester–like assay (CTV) and by cell counting/replating assays. (E) Eµ-Myc cells (no. 107) were transduced with constitutive (pLMS) vectors expressing shHdac3.1659 or shScr, immediately stained with CTV, allowed to expand in culture overnight, and then FACS-sorted to a single population of GFP⁺/CTV⁺ (PacBlue⁺) cells. Cells were serially cultured for up to 5 days and underwent daily assessment of their proliferative capacity by flow cytometry (ie, loss of CTV cells over time; shScr, red line; shHdac3, blue line). Individual plots represent daily flow cytometry analysis and are representative of 3 individual biological experiments. (F) Eµ-Myc lymphoma cells constitutively depleted of Hdac3 were seeded (5 × 10³ cells; day 1) into 24-well plates (shScr vs shHdac3) followed by daily cell counts (individual bars represent days 1, 2, 3, 4, 5), then replated (5× 10³ cells; day 5) and counted daily (n = 3 biological replicates; bars represent days 5, 6, 7, 8, 9). Data are presented as mean ± SEM. (G) Representative histograms demonstrate the percentages of shRNA-expressing (GFP⁺) Eµ-Myc cells on days 3 and 12 of cell counting/replating assay. (H) Eµ-Myc.Cdkn1a^+/+ (no. 107) or Eµ-Myc.Cdkn1a^−/− (no. 152) lymphoma cells were depleted of Hdac3 and cell proliferation was assessed by competitive proliferation assay. Individual bars represent percentages of shRNA-expressing (GFP⁺) Eµ-Myc cells normalized to day 1 (n = 3 biological replicates; individual bars represent days 1, 3, 5, 7, 9, 11). Data are presented as mean ± SEM.