Volume 43, Issue 4, Pages 681-688 (August 2011) The Methyltransferase Set7/9 (Setd7) Is Dispensable for the p53-Mediated DNA Damage Response In Vivo  Stefano Campaner, Fabio Spreafico, Thomas Burgold, Mirko Doni, Umberto Rosato, Bruno Amati, Giuseppe Testa  Molecular Cell  Volume 43, Issue 4, Pages 681-688 (August 2011) DOI: 10.1016/j.molcel.2011.08.007 Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Set7/9 Is Dispensable for the p53 Response Following DNA Damage or Oncogene Activation (A) Immunoblot analysis of Set7/9 levels in tissues derived from mice of the indicated genotypes. Vinculin (vinc.) was used as a loading control. Samples were loaded on the same gel and transferred on the same membrane but are displayed as separate panels for clarity. (B) Immunoblot analysis of Set7/9 levels in MEFs of the indicated genotypes. Vinculin (vinc.) was used as a loading control. Low and high exposures of the film are shown. (C) Cell death following 17 hr of Adr (0.1 or 1 μM) was assessed by trypan blue exclusion. Mean of two independent MEF isolates are shown. Error bars indicate SD. (D) Bidimensional FACS analysis of the cell-cycle distribution of primary MEFs treated with 0.1 μM Adr for 48 hr and pulse-labeled with BrdU before collection. Representative profiles are shown along with the regions used for the analysis reported in the bar graphs. Mean of two independent MEF isolates is shown for Set7/9+/− and Set7/9 −/−. Error bars indicate SD. (E) Twenty-four hours after irradiation (10 Gy), Set7/9 MEFs of the indicated genotypes were labeled with BrdU (33 μM) and then processed for cell-cycle analysis by FACS. The bar graph reports the percentage of BrdU-positive cells of independent MEF isolates of the indicated genotypes. (F) Bidimensional FACS analysis of the cell-cycle distribution of primary MEFs treated with 1 μM Adr for 24 hr and pulse-labeled with BrdU before collection. Data were analyzed as in (D). Molecular Cell 2011 43, 681-688DOI: (10.1016/j.molcel.2011.08.007) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Long-Term Treatments with Adr: Cell Growth and Apoptosis in MEF Cultures Treated with Increasing Concentrations of Adr (A) Growth curves of MEFs, WT or knockouts for Set7/9 from two independent litters, grown in the presence of the indicated concentrations of Adr. The plots report the mean values of triplicate measures. (B) Caspase 3/7 activity measured in MEFs treated as in (A). Bar graphs show the mean values (n = 3) ± SD. (C) Cell death measured in MycER-expressing MEFs of the indicated genotypes, grown in the presence or absence of 400 nM 4-hydroxy-tamoxifen (OHT) for 48 hr. Molecular Cell 2011 43, 681-688DOI: (10.1016/j.molcel.2011.08.007) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Set7/9 Is Dispensable for p53 Posttranslational Modification and p53-Dependent Gene Expression (A and B) Immunoblot analysis of p53 acetylation (Ac-K279 p53) in Set7/9 MEFs following 17 hr of treatment with Adr at the indicated concentrations. Vinculin (vinc.) was used as a loading control. Low and high exposures of the film are shown. A2, B5, B1, B2, and A4 indicate independent MEF preparations. (C) Cellular lysates prepared from MEFs upon Adr treatment were immunoprecipitated (IP) with mono-methyl K372 p53 antibody (Me-K372 Ab). Immunoprecipitates were washed with lysis buffer containing either 0.5 M NaCl (NaCl wash) or with lysis buffer supplemented with 0.1% SDS (SDS wash). (D) Immunoprecipitation experiments performed in the presence of competing peptides used at equimolar concentration to the Me-K372 Ab (1×) or at a 10-fold excess (10×). Cell lysates were prepared from cells treated with Adr for 17 hr. Peptides used were: human p53-peptide-mono-methyl-K372 (Me-Hu), murine p53 (361–376) unmodified (Mu), and p53 (361–376) mono-methyl K369 (Me-Mu). The intensity of each band normalized to the mock IP is reported below each gel scan. (E) Unsupervised hierarchical clustering analysis of the expression level measured by quantitative RT-PCR for selected p53 target genes in MEFs, WT or knockout for Set7/9, following 17 hr of treatment with the indicated concentrations of Adr. Expression levels were normalized to TBP mRNA and reported as expression levels relative to the mock-treated control (gene list and expression values in the Supplemental Information). Molecular Cell 2011 43, 681-688DOI: (10.1016/j.molcel.2011.08.007) Copyright © 2011 Elsevier Inc. Terms and Conditions