Fig. 7
RSL1D1 Recruits p53 to HDM2 to Enhance p53 Ubiquitination. A GST pull-down assays were performed to evaluate the interaction between RSL1D1, p53, and HDM2 in vitro. RSL1D1 was GST-tagged, whereas p53 and HDM2 were His-tagged. B, C Co-IP assay was performed to evaluate the interaction between RSL1D1, p53, and HDM2 in vivo. Lentivirus-transduced HCT116p53−/− cells stably expressing V5-p53 (B) or HCT116p53+/+ cells (C) were harvested and the lysate was immunoprecipitated with anti-V5 (B) or anti-HDM2 (C) antibody. Rabbit IgG was used as a negative control. Input represents 2.5% (B) or 5% (C) of the lysate utilized for IP. D A combination of BiFC and IF assays was performed to evaluate the intracellular co-localization of RSL1D1, p53, and HDM2. After co-transfection with pBiFC-mCherryN159-RSL1D1 and pBiFC-mCherryC160-p53, HCT116 cells were incubated with anti-HDM2 antibody and then FITC-labeled anti-IgG. Red fluorescence indicates an RSL1D1-p53 complex. Green fluorescence indicates HDM2 protein. Yellow color indicates a ternary protein complex comprising RSL1D1, p53, and HDM2. The nuclei were stained with Hoechst (blue). Scale bars: 50 μm. E The protein levels of p53, RSL1D1, and HDM2 were determined in HCT116p53+/+ cells overexpressing truncated RSL1D1 variants or EGFP. The cells were transduced with lentiviruses inducibly expressing genes of interest and treated with 1 μg/mL doxycycline to induce gene expression for 24 h. The protein levels of RSL1D1-NT, RSL1D1-CT, p53, RSL1D1 and HDM2 were determined by western blot analysis. β-actin was used as a loading control. F The levels of ubiquitinated p53 proteins were evaluated in HCT116p53+/+ cells overexpressing truncated RSL1D1 variants or EGFP. The cells were transduced with lentiviruses inducibly expressing genes of interest and treated with 1 μg/mL doxycycline to induce gene expression for 24 h, followed by an additional treatment with 25 μM MG-132 for 6 h. DO-1 was used for IP and western blot analyses of the ubiquitinated or non-ubiquitinated p53 protein. The protein levels of RSL1D1-NT, RSL1D1-CT, p53, RSL1D1, and HDM2 in the input lysate were determined by western blot analysis and β-actin was used as a loading control. G A model showing the interaction between RSL1D1, p53, and HDM2, which can be competitively destroyed by overexpression of either RSL1D1-NT or RSL1D1-CT