Figure 1.

PHD3 stabilizes p53. A, overexpression of PHD3 increased p53. CCD841 and RKO cells were transfected with control or myc-PHD3 plasmid. After 24 h, the cells were harvested for Western blotting. B, overexpression of PHD3 had little effect on mRNA levels of TP53. The cells were treated as in A. C, knockdown of PHD3 decreased p53. The cells were transfected with control or PHD3 siRNA oligonucleotides. After 48 h, the cells were harvested. D, RKO and CCD841 cells were transfected with control or siPHD3 oligonucleotides as indicated. After 48 h, the cells were harvested for determination of TP53 mRNA level by qPCR. E, CCD841 and RKO cells were transfected as indicated. After 24 h, cycloheximide (10 μg/ml) was added to inhibit new protein synthesis, and the cells were harvested at different time intervals. The right panel shows the relative p53 level at different time point. F, CCD841 cells were transfected with control or siPHD3 oligonucleotides. After 48 h, the cells were treated with cycloheximide (10 μg/ml) for different time intervals. The right panel shows the relative p53 level. G, proteins extracted from small intestinal and colonic epithelial cells of Phd3^F/F and Phd3^IEC-KO mice were subjected to Western blotting. The p53(DO-7) antibody was used to detect mouse p53. H, the cells were transfected with control or myc-PHD3 plasmid as indicated. After 24 h, the cells were harvested. The Western blots were quantified using ImageJ, and the results are from one experiment. IB, immunoblotting; ns, no significance.