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. 2014 Mar 23;5(8):2116–2130. doi: 10.18632/oncotarget.1845

Figure 5. Relative expression of endogenous P63 isoforms in cells lines treated with DNA damaging or P53 inducing agents.

Figure 5

Experiments were conducted in the indicated cell lines with known P53 status (wild type, mutant or null, as indicated). Cells were treated with Doxorubicin (DXR), 5-FluoroUracil (5FU) or Nutlin-3a (Nutlin) for 16 hours, as described in Materials and Methods. B2M and GAPDH served as reference genes. Three biological replicates were performed. Measurements of the endogenous levels of ΔN-P63 or TA-P63 mRNAs in P53 wild type A) or mutant/null cells B). For each cell line the graph indicates the relative expression changes and is normalized over the most abundant isoform transcript, set to 1 for the DMSO (mock solvent condition, broken line). C) The endogenous levels of the P53 target gene P21 were measured as a positive control of the efficacy of the treatments. Values are indicated as fold change relative to DMSO treated cells (set to 1 and indicated by broken line). D) ΔN-P63 protein levels were determined by Western blot in JHU-029 cells after treatment with DXR, 5FU or Nutlin. GAPDH was used as loading control. Immunoreactive bands specific for the ΔN-P63α or ΔN-P63β isoforms are indicated with black arrows. E) Western blot experiment demonstrating the over-expression of P53 after transfection and its effect on endogenous ΔN-P63α or ΔN-P63β isoforms in JHU-029 cells. GAPDH was used as loading control.