Fig 2: The signaling of AC3RL-induced senescence in HAECs. HAECs were incubated with PBS (control, ctrl), AC3(-)L (25 µg/mL), AC3RL (25 µg/mL), AC3RL + siFBXO31 (20 nM), AC3RL + NAC (5 mM), or AC3RL + SM (3 µM) for 24 h (A–C). Then, the protein expressions of p53 and p21 (A) or FBXO31 and p-MDM2 (B,C) were determined using Western blotting. Actin was used as an internal control. For the quantitative analyses, n = 3 per group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. AC3RL.

Fig 3: AC3RL induced cell senescence via FBXO31. HAECs were incubated with PBS (control, ctrl), AC3(-)L (25 µg/mL), AC3RL (25 µg/mL), and/or AC3RL + siFBXO31 (20 nM) for 24 h. (A) SA-ß-Gal staining in HAECs from each treatment group. (B) Intracellular reactive oxygen species (ROS) production in HAECs. (C) Immunofluorescence staining showing ?H2AX foci in HAECs from each treatment group. Positively stained cells were quantified and are represented as a proportion of total cells. For the quantitative analyses, n = 3 per group. ** p < 0.01 vs. control; # p < 0.05, ## p < 0.01 vs. AC3RL.

Fig 4: Depletion of FBXO31 regulates ERK and AKT activity and promotes cell growth in prostate cells(A) Parental and FBXO31 KO RWPE1 cells were treated with vehicle or the DUSP6 inhibitor BCI for the indicated days, and cell viability was determined by ATP-Glo assay. Graph was generated using Prism 8 (mean ± SD; n = 8).(B) VCAP cells stably transduced with a retroviral system expressing doxycycline-inducible FBXO31 or a control vector were treated with doxycycline for the indicated times. WCLs were immunoblotted as indicated.(C) RWPE1 cells were treated with various concentrations of BCI for 6 h and then analyzed by immunoblotting for the indicated proteins.(D) FBXO31 wild-type or KO RWPE1 cells were cultured in medium without EGF for the indicated times (withdrawal [W/D]) and then harvested and analyzed by immunoblotting.(E) FBXO31 wild-type or KO RWPE1 cells were cultured in regular medium or in medium without EGF (W/D). Where indicated, cells were treated with the DUSP6 inhibitor BCI. GTP-bound RAS was quantified using RAF1 Ras-binding domain (RBD) pull-down assay. Western blotting images are representative of 3 independent experiments, and the intensity of bands was quantified by ImageJ. The columns represent the relative levels of RAS-GTP as normalized to the levels of total RAS protein (mean ± SD; n = 3 independent experiments)(F) FBXO31 wild-type or KO RWPE1 cells were cultured in regular medium or medium without EGF. Relative cell viability was measured by ATP-Glo assay. Graph was generated using Prism 8 (mean ± SD; n = 8).

Fig 5: FBXO31 mediates DUSP6 degradation and regulates ERK signaling(A) 293H cells were transfected with plasmids expressing the indicated proteins. Cells were treated with the proteasome inhibitor MG132 for 4 h. Cell lysates were then immunoprecipitated with an anti-FLAG resin, and the immunoprecipitates were probed with the indicated antibodies.(B) Parental RWPE1 cells and two FBXO31 KO RWPE1 clones were subjected to western blot analysis using the indicated antibodies.(C) DU145 cells were transfected with either non-targeting control siRNA or siRNA against FBXO31. Cells were then treated with cycloheximide (CHX) for the indicated times. Protein extracts were immunoblotted for the indicated proteins.(D) 293H cells were transfected with hemagglutinin (HA)-tagged DUSP6, FLAG-tagged FBXO31, or its F-box domain deletion mutant (?Fbox) as indicated. After immunopurification (IP) with anti-FLAG resin, in vitro ubiquitylation of DUSP6 was performed in the presence or absence of E1, E2s (UbcH3 and ubcH5), and ubiquitin (ub). Samples were analyzed by immunoblotting with an anti-DUSP6 antibody. Immunoblots of whole-cell lysates (WCLs) are shown at the bottom.(E) MEFs stably expressing a control shRNA or a shRNA against FBXO31 were serum-starved overnight, then stimulated with EGF (100 ng/mL). Samples were collected at the indicated time points for immunoblotting analysis.(F) The expression levels of ERK signaling-regulated genes in prostate patient samples with higher (top 25%) or lower (bottom 25%) FBXO31 mRNA levels are depicted as a heatmap. RNA sequencing (RNA-seq) data were obtained from the TCGA dataset of human prostate adenocarcinoma (Cancer Genome Atlas Research Network, 2015).