Fig. 4

NAT10 stabilizes mutant p53 by counteracting Mdm2 action. a LO2, HepG2, MHCC-97H and MHCC-97 L cells were harvested and fractionated. Fractions were then immunoblotted with the indicated antibodies. (C, cytoplasmic; N, nuclear) b HCC cells were seeded on coverslips and stained with anti-NAT10 and anti-p53 antibodies. Nuclei were stained with DAPI. Fluorescence images were photographed under confocal microscopy. c Huh7 cell lysates were immunoprecipitated with control IgG or anti-NAT10 antibodies. The immunoprecipitates were subsequently immunoblotted with the indicated antibodies. d Huh7 cells were transfected with the indicated siRNAs. Seventy-two hours later, the total proteins were analyzed by western blotting for the indicated proteins. e Huh7 cells were transfected with the indicated siRNAs and treated with MG132 for 4 h before harvest. The whole cell lysates were analyzed by western blotting for the indicated antibodies. f Huh7 cells were transfected with the indicated plasmids. Forty-eight hours later, cells were harvested after MG132 treatment, and the whole cell lysates were analyzed by western blotting for the indicated antibodies. (NAT10 GE: NAT10 mutant lacking acetyltransferase activity; NAT10 D5: NAT10 mutant lacking ubiquitin ligase activity) g Huh7 cells were transfected with the indicated plasmids. Forty-eight hours later, cells were harvested and lysed. The whole cell lysates were analyzed by western blotting for the indicated antibodies. h Huh7 cell lysates were immunoprecipitated with control IgG or anti-NAT10 antibodies. The immunoprecipitates were subsequently immunoblotted with the indicated antibodies. i Huh7 cells transfected with the indicated siRNAs were plated in 96-well plates, and cell proliferation was then quantified at the indicated time points. j MHCC-97 L cells transfected with the indicated siRNAs or vectors were plated in 96-well plates, and cell proliferation was then quantified at the indicated time points as described in Methods