Fig. 1

Acute myeloid leukemia model with inducible reactivation of the partial LOF variant p53^E177R. a Experimental outline for in vivo generation of AML by retroviral transduction of fetal liver cells from embryos expressing CreER^T2 together with constitutive p53 knockout (KO) or Cre-inducible partial LOF p53 variant E177R (LSL-E177R). Leukemia, that developed after transplantation of transduced FLCs into irradiated wild-type recipient mice, was further propagated ex vivo for cell culture studies or transplanted into secondary recipients for in vivo reactivation experiments. b Scheme depicting CreER^T2-mediated excision of the floxed stop cassette (LSL), which silences p53 expression in the LSL-E177R allele. Shown are primers A-D used for genotyping and validating Cre-mediated recombination of the Trp53 gene locus. c PCR genotyping: switch from the A-B to C-D PCR product reveals efficient recombination in Trp53^{LSL-E177R/LSL-E177R}; Rosa26^CreERT2 AML cells upon 48 h treatment with 1 μM 4OHT. d Immunoblot demonstrating induction of p53 and p21/Cdkn1a protein expression upon 4OHT treatment of LSL-E177R; CreER^T2 (short: LSL) AML cells. β-actin is shown as a loading control. e Quantitative reverse transcription PCR for Trp53 and p53 target gene induction in LSL-E177R; CreER^T2 and KO; CreER^T2 AML cells 48 h after treatment with 4OHT. Data were normalized to Actb and mock treatment. f Chromatin immunoprecipitation analysis of p53 at the indicated target gene promoters 48 h after 4OHT or mock treatment. Chromatin samples were immunoprecipitated with α-p53 antibody (FL393) or IgG as background control. Shown is DNA binding as % input chromatin. e-f Shown are mean ± SD; datapoints represent biological replicates (n = 3); reported are 2way ANOVA P-values for the interaction between the two independent variables treatment and genotype and P-values for indicated pairwise comparisons (Sidak’s multiple comparisons test)