Stabilization of p73 after treatment with ETP is insufficient in Kpm/Lats2-knockdown cells. (A) Western blotting analysis with anti-p73 in whole-cell lysate of control KG-1a cells without treatment of ETP (lane 1) or with treatment of 0.03 μg/mL ETP for 3 days (lane 2), Kpm/Lats2-knockdown KG-1a cells without treatment of ETP (lane 3), or with treatment of 0.03 μg/mL ETP for 3 days (lane 4), and p73-knockdown KG-1a cells without treatment of ETP (lane 5) or with treatment of 0.03 μg/mL ETP for 3 days (lane 6). Twice, independent experiments were performed and representative data are shown. (B) Expression of p73 mRNA in Kpm/Lats2-knockdown KG-1a or control cells treated with 0.03 μg/mL ETP for 3 days was measured by real-time PCR analysis. Data normalized to gapdh are shown as mean plus or minus SD in scale that the value for KG-1a wild-type cells with the same treatment is 1. The analyses were performed in triplicate independently twice and representative data are shown as mean plus or minus SD. (C) Immunofluorescence staining for p73 and DAPI. Kpm/Lats2-knockdown KG-1a cells and control cells treated with 0.1 μg/mL ETP were stained with anti-p73 Ab or DAPI and observed with a fluorescence microscope at the magnification of ×100 or ×600. Four independent experiments were performed and representative data are shown. (D) ChIP assay. ChIP assay was performed as described in “Chomatin immunoprecipatation assay.” p73 was recruited to PUMA gene promoter in control cells but not in Kpm/Lats2-knockdown cells after the treatment with 0.03 μg/mL ETP for 60 hours. Two independent experiments were performed and representative data are shown.