Fig. 7: PDCD11 negatively regulates p53 by recruiting p53 to HDM2.
Combinational BiFC-IF assay was performed to investigate the intracellular colocalization of PDCD11, p53, and HDM2. HCT116p53-/- cells co-transfected with pBiFC-mCherryN159-PDCD11 and pBiFC-mCherryC160-p53 were subjected to IF assay using anti-HDM2 (A), whereas HCT116p53+/+ cells co-transfected with pBiFC-mCherryN159-PDCD11 and pBiFC-mCherryC160-HDM2 were immunostained by anti-p53 (B). Red fluorescence indicates a PDCD11-p53/PDCD11-HDM2 complex and green fluorescence indicates endogenous HDM2/p53. Yellow color indicates the colocalization of the three proteins. The nuclei were stained to blue. Scale bars: 10 µm. C–H All the lentivirus-transduced HCT116p53+/+ cells were treated with Doxy to induce expression of Luc, FLAG-PDCD11 (1-1032), or His-NLS-PDCD11 (1033-1431) in HCT116p53+/+ cells. C PLA was performed to investigate PDCD11 (1-1032)-p53, PDCD11 (1033-1431)-HDM2, PDCD11-p53/HDM2, and p53-HDM2 interactions (red) in the cells treated with MG-132 (20 µM, 8 h). The nuclei were stained to blue. Scale bars: 25 µm. D, E Ubiquitinated and non-ubiquitinated p53 was immunoprecipitated using anti-p53 (DO-1) from the cells treated with MG-132 (20 µM, 8 h). Anti-ubiquitin (P4D1) and anti-p53 (DO-1) were used to detect ubiquitinated and non-ubiquitinated p53, respectively. The levels of FLAG-PDCD11 (1-1032)/His-NLS-PDCD11 (1033-1431), p53, and HDM2 proteins in the input cell lysate were determined by western blotting and GAPDH was used as a loading control. F, G Western blot analyses were performed to determine the levels of FLAG-PDCD11 (1-1032)/His-NLS-PDCD11 (1033-1431), p53, and HDM2 proteins in the cells expressing Luc, FLAG-PDCD11 (1-1032), or His-NLS-PDCD11 (1033-1431). GAPDH was used as a loading control. H A model showing the formation of PDCD11-p53-HDM2 complex, which can be competitively destroyed by overexpression of either PDCD11 (1-1032) or PDCD11 (1033-1431).
