Fig 1: USP47 modulated OC cell viability. (A and B) Cell cycle distribution of MG-63 and U2OS cells with or without USP47 knockdown via specific USP47-siRNAs (n = 5). (C) Apoptosis rates in MG-63 and U2OS cells transfected with siRNAs (n = 5). (D) Cleaved-caspase3 protein levels demonstrating apoptosis induced by FEN1 or USP47 knockdown. (E and F) Comet assay and γH2AX protein levels showing DNA damage after USP47 knockdown in MG-63 and U2OS cells. (Scale bar: 100 μm, n = 5). Results are the mean ± SD. **P < 0.01, and ***P < 0.001.
Fig 2: FEN1 modulated OS cell viability. (A and B) Cell cycle distribution of MG-63 and U2OS cells with or without FEN1 knockdown using specific siRNAs (n = 5). (C) Apoptosis rates of MG-63 and U2OS cells transfected with FEN1-siRNAs (n = 5). (D and E) Comet assay and γH2AX protein levels demonstrating DNA damage after FEN1 knockdown in MG-63 and U2OS cells. (Scale bar: 100 μm, n = 5). (F) USP47 was predicted to bind to FEN1 using the BioGrid database. (G) The combination of USP47 and FEN1 was verified by HDock software. Results are the mean ± SD. ***P < 0.001.
Fig 3: Effects of rotenone on USP47 and molecular docking analysis. (A) 293T cells treated with rotenone for the indicated times; FEN1 protein was detected as shown. (B) Co-IP analysis showed the interaction between FEN1 and USP47 when treated with rotenone. (C) Rotenone was predicted to bind to USP47 using molecular docking. (D) The combination of rotenone and USP47 was verified by Biacore analysis. (E) U2OS cells were treated with rotenone and the Ub-VME probe was added. Samples were subsequently analyzed by Western blotting using anti-USP47 antibody to detect the Ub-USP47 conjugate formation. (F) 293T cells were co-transfected with Myc-FEN1, Flag-USP47, and treated with or without rotenone; polyubiquitination analysis of FEN1 was carried out. Results are the mean ± SD. *P < 0.05, and **P < 0.01.
Fig 4: Rotenone suppressed the USP47/FEN1 pathway in vivo. (A) Tumor growth of subcutaneously implanted mice was measured every 3 days (n = 5). (B) Tumor weight was measured. (C) The survival rate of tumor-bearing mice (time to 20 mm in maximum diameter) was determined by Kaplan-Meier analysis; statistical differences were analyzed with Log rank test and survival. Scale bar: 100 μm. (D) Representative IHC images of USP47 and FEN1 in xenograft tissues. (E) Protein expression of USP47 and FEN1 in xenograft tissues. (F) Working model illustrating Rotenone inhibited OS cell growth through USP47-induced decreases in FEN1 stability and DNA integrity. Results are the mean ± SD.*P < 0.05, and ***P < 0.001.
Fig 5: USP47 interacted with and deubiquitinated FEN1. (A) FEN1 protein levels with USP47 siRNA or expression plasmid transfection. (B) Confocal microscopy was used to clarify the co-location of FEN1 and USP47. (Scale bar: 50 μm, n = 5). (C) Co-IP analysis was performed to test the interaction between FEN1 and USP47. (D and E) 293T cells were co-transfected with Myc-FEN1, Flag-USP47, and Flag-USP47 mutant plasmids, and polyubiquitination analysis of FEN1 was carried out. (F) γH2AX and cleaved-caspase 3 protein levels after USP47 knockdown in FEN1 low/high-expression OS cells. (G) Cell cycle distribution of MG-63 and U2OS cells treated with indicated conditions (n = 5). (H) Apoptosis rates in MG-63 and U2OS cells transfected with indicated plasmids (n = 5). (I) Comet assay showing DNA damage after treatment. (Scale bar: 200 μm, n = 5). (J and K) USP47 and FEN1 protein (n = 46 for OS, n = 38 for control) and mRNA (n = 7) expression in human OS and control group tissues. Scale bar: 50 μm (Red) or 20 μm (Black). (L) Correlation between USP47 and FEN1 expression based on the GEPIA database. Results are the mean ± SD. ***P < 0.001.
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