Hdac1 and Hdac2 dosage-dependent requirement for p53 inactivation. (A) Western blot of protein lysates from tumors with indicated genotypes using Hdac1, Hdac2, p19^Arf, and p53 antibodies. α-tubulin served as loading control. (B) Summary of p53 staining intensity, indicative for stabilized/mutant p53, of a tissue micro-array containing Hdac1^+/Δ;Hdac2^Δ/Δ (n = 6), Hdac1^Δ/Δ (n = 8), and Hdac1^Δ/Δ;Hdac2^+/Δ (n = 36) tumors, revealed a significant decrease of mutant p53 in Hdac1^Δ/Δ;Hdac2^+/Δ tumors (Fisher test, P = .0038; bottom table). (C) Western blot analysis of Hdac1^+/Δ;Hdac2^Δ/Δ, Hdac1^Δ/Δ and Hdac1^Δ/Δ;Hdac2^+/Δ lymphoma cell lines as well as wild-type thymocytes for expression of p53 and p19^Arf. Tubulin served as a loading control (top). p53 DNA binding domain missense mutations (F130S) in Hdac1^Δ/Δ and Hdac1^+/Δ;Hdac2^Δ/Δ tumor cell lines. p53 sequence of wild-type thymocytes served as reference (bottom). (D) Western blot analysis of Nutlin-3–treated Hdac1^Δ/Δ;Hdac2^Δ/+ lymphoma cell line for expression of p53. Human MOLT-3 T-ALL cell line harboring wild-type p53 and a murine p53^Δ/Δ T-cell lymphoma served as controls. Tubulin served as a loading control. Nutlin-3 treatment resulted only in wild-type p53 bearing lymphoma cell lines in loss of viability, as determined by cell-titer blue assay (lower panel) and visual inspection of cell cultures (right panel; magnification 200×). (E) Western blot analysis of protein lysates of p53^Δ/Δ, Hdac1^Δ/Δ, and Hdac1^Δ/Δ;Hdac2^+/Δ tumor cell lines treated with ionizing radiation (6 Gy) for p53 and p19^Arf. Hdac1^Δ/Δ;Hdac2^+/Δ tumor cell lines showed radiation-induced p53 in contrast to an Hdac1^Δ/Δ lymphoma cell line expressing mutant p53. (F) Western blot analysis (top panel) of p53 protein levels in 1-week-old WT and Hdac1^Δ/Δ;Hdac2^Δ/+ thymocytes, 6 hours after mock or 6 Gy γ-irradiation. Tubulin served as loading control. Bottom panel shows quantification of p53 levels relative to tubulin. Error bars indicate standard deviations of thymocyte cultures isolated from 3 independent mice per genotype. (G) Percentage of viable cells in 3 independent WT and Hdac1^Δ/Δ;Hdac2^Δ/+ thymocyte cultures 24 hours after indicated doses of γ-irradiation. WT, wild type.