NPM-ALK induces p16INK4a promoter de-methylation. (A) Top panel: chromatin immunoprecipitation analysis of p16INK4a promoter H3K27me3 levels in WT MEFs infected with the indicated retroviruses. Precipitated DNA was amplified by qPCR using primers located in the p16INK4a promoter region. Enrichments are presented as percentage of total input DNA. Bottom panel: Q-PCR analysis of p16INK4a mRNA levels in the same cells, expressed as fold increase with respect to the day 1 control vector. (B) Growth curves of WT MEFs infected with a control lentivirus or lentiviruses expressing JunB-specific shRNAs, in the presence or absence of NPM-ALK, as indicated. Values are expressed relative to day 1 for each sample. *P < .05 with respect to control cells infected with a control vector. (C) Western blotting of p16INK4a, JunB, and NPM-ALK expression in the same cell samples as in panel B, 4 days after infection. Tubulin was used as a loading control. p16INK4a protein levels were calculated by densimetric analysis and normalized to control (Ctrl.) and tubulin band intensities. (D) Western blotting of Jmjd3 and p16INK4a expression levels in WT MEFs infected with NPM-ALK or control viruses. p16INK4a protein levels were calculated as in panel C. (E) Growth curves of WT and Jmjd3 null MEFs infected as indicated. Values are expressed relative to day 1 for each sample. (F) Western blotting of p16INK4a and NPM-ALK expression levels in WT and Jmjd3-null MEFs in the same cell samples as in panel E, 4 days after infection. p16INK4a protein levels were calculated as in panel C. (G) Left panel: Western blot analysis of STAT3, p16INK4a, and tubulin expression at different times (as indicated) in TS cells treated with doxocyclin to induce shRNA interference targeting STAT3. Right panel: p16INK4a mRNA quantification by qPCR analysis with specific primers. Data were standardized with ribosomal RNA and normalized against control untreated cells.