Fig 2: RNF157 interacts with p53 and promotes the ubiquitination of p53. (A) HLE-B3 cells were co-transfected with Flag-RNF157 and Myc-p53 plasmids and then treated with MG132 (10 µM) for 6 hours before immunoprecipitation (IP) followed by western blot analysis. IgG served as a negative control. (B) Representative fluorescence images of RNF157 and p53 in HLE-B3 cells. Scale bar = 10 µm. (C) HLE-B3 cells were co-transfected with HA-ubiquitin, Myc-p53, and Flag-RNF157 or empty plasmids for 36 hours. Cells were then treated with 10 µM MG132 for 6 hours, followed by IP and Western blot analyses. (D) HLE-B3 cells were co-transfected with HA-ubiquitin, Myc-p53 plasmids, and RNF157 siRNA or NC siRNA for 48 hours and then treated with 10 µM MG132 for 6 hours. IP and Western blot analyses were performed using the indicated antibodies.

Fig 3: The E3 ligase RNF157 facilitates p53 degradation through the proteasome pathway. (A) The proteins were enriched in the positive regulation of intrinsic apoptotic signaling pathway. (B, C) Influence of RNF157 on endogenous or exogenous p53 protein expression. (D) The qRT-PCR was performed to detect the effects of exogenous RNF157 on p53 mRNA expression. The values shown are mean ± standard deviation (n = 3). ns means not significant. (E) Effect of RNF157 knockdown on the p53 protein level. (F) HLE-B3 cells were transfected with empty vector or Flag-RNF157 vector for 36 hours and then were treated with 10 µM MG132 for 6 hours before Western blotting. (G) HLE-B3 cells transfected with empty vector or Flag-RNF157 vector were treated with CHX (25 µg/mL) for the indicated durations and analyzed by Western blotting. (H) HLE-B3 cells transfected with NC siRNA or RNF157 siRNA were treated with CHX (25 µg/mL) for the indicated duration before Western blot analysis. * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig 4: RNF157 knockdown sensitives HLE-B3 cells to H2O2-induced apoptosis. (A, B) RNF157 siRNA transfection efficiency detected by qRT-PCR and western blotting. The values shown are mean ± standard deviation (n = 3). ** P < 0.01, *** P < 0.001. ns means not significant. HLE-B3 cells were transfected with RNF157 siRNAs or NC siRNA for 24 hours and then were treated with 200 µM H2O2 for another 24 hours. (C, D) Western blotting and quantification of caspase-3 proteins in treated HLE-B3 cells. (E, F) Flow cytometry analysis of cell apoptosis after the indicated treatment. The values shown are mean ± standard deviation (n = 3). * P < 0.05, *** P < 0.001.

Fig 5: The p53 mediates the effects of RNF157 on H2O2-induced cell apoptosis. (A, B) HLE-B3 cells were transfected with Flag-RNF157 plasmid, Myc-p53 plasmid, or empty vector, caspase-3 activity was measured by Western blotting, and the apoptosis rate was tested via flow cytometry analysis. The values shown are mean ± standard deviation (n = 3). * P < 0.05, ** P < 0.01. (C) Knockdown efficiency of p53 siRNAs in HLE-B3 cells. (D, E) Effects of double knockdown of RNF157 and p53 on the activation of caspase-3 and the cell apoptosis rate upon H2O2 treatment. The values shown are mean ± standard deviation (n = 3). * P < 0.05, ** P < 0.01.