Dhx9 suppression activates proapoptotic p53 signaling. (A) p53 transcriptional targets are elevated on Dhx9 suppression. The distribution of fold changes between Arf^−/−Eµ-myc/Bcl2 lymphomas expressing Rluc.713 or Dhx9.1241 was compared for p53 target genes²⁴  and non-p53 target genes. n = 3; Kolmogorov-Smirnov test, P = .0005. (B) Validation of microarray results by quantifying p53 transcriptional targets by quantitative reverse transcription-polymerase chain reaction. Values are set relative to β-actin. Error bars represent ± standard error of the mean; n = 6. (C) Heat map and clustering analysis of p53 targets in Arf^−/−Eμ-myc/Bcl-2 lymphomas expressing Rluc.713 or Dhx9.1241. Cells were harvested 5 days after infection, and 3 independent biological replicates were used for each shRNA. The expression relative to per gene mean and standard deviation (z-score) is rendered in a red-black-green pseudo color scheme for p53 targets.²⁴  Hierarchical clustering indicates p53 transcriptional target efficiently segregate lymphomas expressing Rluc.713 from those expressing Dhx9.1241. (D) Immunoblot analysis of cell extracts prepared from Arf^−/−Eμ-myc/Bcl-2 and p53^−/−Eμ-myc/Bcl-2 lymphoma cells. Cells were infected with MLS constructs expressing the indicated shRNAs (top) and probed with antibodies to the protein targets indicated to the left. Quantitation of the average fold p53 induction in response to Dhx9 suppression from 3 independent experiments. (E) Immunoblot analysis of cell extracts prepared from Arf^−/−Eμ-myc/Bcl-2 lymphoma cells infected with MLS constructs expressing the indicated shRNAs.