Fig. 6

JMJD6 overexpression reduces p53 and p19ARF levels. a Immunoblotting of NMuMG cells with ectopic expression of JMJD6 or LacZ control shows decreased protein levels of p53 and p19ARF in cells with or without Myc induction. b Cells were stimulated with 4-OHT to induce Myc and then treated with 100 mM etoposide for 4 and 8 h to analyze p53 induction. Immunoblots show lower levels of p53 or Ser18-phosphorylated p53 as well as p19ARF and p21 in cells expressing JMJD6. Intensity of each band normalized to β-actin was obtained using ImageJ software. c Overexpression of p19ARF in the presence of JMJD6 restores Myc-induced cell death. NMuMG cells expressing MycER™ together with JMJD6, LacZ, or JMJD6 plus p19ARF were treated with 4-OHT (MycON) or ethanol (MycOFF) for 24 h and placed in serum-free media for another 24 h. Percentage of dead cells was measured with CytoTox-Glo reagent. Black bars—MycON, open bars—MycOFF. d Western blot analysis of NMuMG cells with ectopic overexpression of p19ARF. e RT-qPCR analysis shows that overexpression of JMJD6 in NMuMG cells inhibits p19ARF transcription. Black bars—NMuMG cells expressing LacZ control, open bars—cells expressing wild-type JMJD6. f Knock-down of endogenous JMJD6 with three different shRNAs increases mRNA levels of p19ARF