Fig. 2
RSL1D1 Negatively Regulates the Level of Nuclear p53 Protein in an HDM2-Dependent Manner. Cells were transfected with siRSL1D1 to downregulate RSL1D1. For overexpression of RSL1D1, cells were transduced with lentiviruses inducibly expressing RSL1D1 and then treated with 1 μg/mL doxycycline for 72 h to induce expression. A The mRNA levels of RSL1D1, p53, HDM2, p21, and PUMA were determined by qRT-PCR analysis in RSL1D1-downregulated HCT116p53+/+ and HCT116p53−/− cells. GAPDH was used as an internal control to normalize the values. The normalized values of siNC-transfected HCT116p53+/+ cells were set to 1. B The protein levels of RSL1D1, p53, HDM2, p21, and PUMA were determined by western blot analysis in RSL1D1-downregulated HCT116p53+/+ and HCT116p53−/− cells. β-actin was set as a loading control. C The mRNA levels of RSL1D1, p53, HDM2, p21, and PUMA were determined by qRT-PCR in RSL1D1-overexpressed HCT116p53+/+ and HCT116p53−/− cells. GAPDH was used as an internal control to normalize the values. The normalized values of EGFP-overexpressed HCT116p53+/+ cells were set to 1. D The protein levels of RSL1D1, p53, HDM2, p21, and PUMA were determined by western blot analysis in RSL1D1-overexpressed HCT116p53+/+ and HCT116p53−/− cells. β-actin was used as a loading control. E The levels of nuclear and cytoplasmic RSL1D1, p53 and HDM2 proteins were determined by western blot analysis in RSL1D1-downregulated HCT116p53+/+ and HCT116p53−/− cells. HDAC1 and β-actin were set as internal controls for nuclear and cytoplasmic proteins, respectively. F The levels of RSL1D1, p53, and HDM2 proteins were determined by western blot analysis in RSL1D1-downregulated HCT116p53+/+ cells treated with Nutlin-3 (40 μM, 12 h). β-actin was used as a loading control. A, C Data are represented as mean ± SD. Student’s t test. *P < 0.05 and **P < 0.01 denote significant difference