Fig 1: SPOP mutants impair DNA end resection and chromosomal stability.(A to I) PC-3 cells infected with EV or lentivirus expressing SPOP mutant F102C or F133V were treated with IR. At each time point after IR, the cells were harvested for IFC with antibodies for SHLD2 (B), RPA2 (D), and BRCA1 (G). The average 53BP1 foci number (B, E, and H) and foci density (C, F, and I) in each cell were quantified. Scale bars, 10 µM. Data are presented as means ± SEM of more than 300 cells from three biological replicates. ***P < 0.001. (J and K) Comparison of CNA burden and nonsynonymous single-nucleotide variation (nsSNV) frequencies per genome between SPOP mutant patients (red bars) and SPOP WT patients (green bars) (J). Comparison of frequencies of structural variations between SPOP mutant patients (red bars) and SPOP WT patients (green bars) (K). P values were calculated using two-sided Wilcoxon rank sum test. (L and M) Control (sgControl) or 53BP1 KO (sg53BP1) BPH1 cells infected with lentivirus expressing EV or SPOP F133V mutant were treated with vehicle (DMSO) (L) or CPT (1 µM) for 24 hours (M). Cells were harvested for karyotyping, and chromosome breaks of more than 300 cells from three biological replicates in each group were counted and quantified.
Fig 2: Structural basis for the interaction of 53BP1 with SPOP and SPOP phosphorylated at serine-119.(A) Backbone NMR signal assignments of 53BP11606–1656. Shown is the 1H-15N heteronuclear single quantum coherence (HSQC) spectrum of 15N- and 13C-labeled 53BP1 (residues 1606 to 1656) with assigned signals. In blue are residues remaining from the expression plasmid (G3, H4, and M5) and two C-terminal residues (G57 and W58) added for protein quantification by ultraviolet light absorption. (B) Representative 1H-15N HSQC spectra of the interaction of 15N-labeled 53BP1 (residues 1606 to 1656) with unlabeled SPOP MATH domain. The 53BP1:SPOP molar ratio is 1:2. The spectra were recorded at 4°C. (C) Representative 1H-15N HSQC spectra of the interaction of 15N-labeled SPOP MATH domain with nonlabeled 53BP1 (residues 1606 to 1656). The SPOP:53BP1 molar ratio is 1:6. The spectra were recorded at 25°C. (D) Surface and cartoon representation of the x-ray crystal structure of the SPOP MATH domain in complex with a 53BP1 SBM synthetic peptide (residues 1636 to 1650). SPOP residues for which NMR signals are affected by 53BP1 binding (see C) are highlighted in red. (E) Details of the SPOP-53BP1 interface in the crystal structure. The blue spheres represent water molecules. The yellow dashes represent hydrogen bonds. (F) Comparison of the SPOP-53BP1 and SPOP S119D-53BP1 crystal structures highlighting the change in conformation in SPOP S119D that brings D119 in contact with 53BP1. SPOP WT and S119D are shown in gray and blue, respectively. Key residues in the vicinity of S119 and D119 in the two structures are labeled. Hydrogen bonds involving S119 and D119 are shown as yellow dashes. (G) Effects of S119D and S119N mutations on SPOP structure. Shown are the 1H-15N HSQC spectra of SPOP MATH mutants S119D and S119N overlaid to that of WT SPOP MATH.
Fig 3: Estrogen potentiates SPOP-mediated degradation of ERa. (a) Estrogen enhances the SPOP-ERa interaction. FH-ERa and Myc-SPOP constructs were co-transfected into 293T cells. After 24 h, cells were treated with the vehicle ethanol (EtOH,-) or 10 nM 17ß-estradiol (E2) for 4 h before cell lysates were prepared for co-IP and WB analyzes. (b) Estrogen enhances SPOP-mediated ERa degradation. The 293T cells were transfected with the indicated constructs. A small amount of Myc-SPOP constructs was used in transfection. After 24 h, cells were treated with the vehicle ethanol (EtOH) or 10 nM 17ß-estradiol (E2) for 4 h before cells lysates were prepared for WB analyzes. The density of ERa was determined by normalizing to actin (loading control) first and then to the normalized value in mock-treated cells. (c) Knockdown of SPOP attenuates estrogen-induced degradation of ERa. Ishikawa cells were transfected with control or SPOP-specific siRNA. After 48 h, cells were then treated with the vehicle ethanol (EtOH,-) or 10 nM 17ß-estradiol (E2) for 4 h before cell lysates were prepared for WB analyzes. (d) Estrogen potentiates SPOP-induced polyubiquitination of ERa. The 293T cells were transfected with the indicated constructs. After 24 h, cells were treated with the vehicle ethanol (EtOH,-) or 10 nM 17ß-estradiol (E2). Cells were then treated with MG132 for 4 h before cell lysates were prepared for IP and WB analyzes. (e) Ishikawa cells lines that stably transfected with control, SPOP-WT or SPOP mutants constructs were treated with 10 nM 17ß-estradiol (E2) for 24 h. The mRNA level of ERa target gene GREB1 was measured by qRT-PCR. The mRNA level of GAPDH was used for normalization. The mean values (S.D.) of three independent experiments are shown. **indicates statistical significance (**P<0.01). (f, g) Differential effects of estrogen on the protein level of ERa-WT and the SPOP degradation-resistant mutant (ERa-M4). The 293T cells were transfected with FH- ERa-WT or M4 mutant construct. After 24 h, cells were treated with vehicle ethanol (EtOH,-), 10 nM 17ß-estradiol (E2), 10 nM Tamoxifen (Tam), and 10 nM Fulvestrant (Ful) for 4 h before cell lysates were prepared for WB analyzes
Fig 4: The S/T-rich motifs in ERa are degrons recognized by SPOP. (a) Schematic representation of wild-type ERa protein with the upper contiguous Ser/Thr residues indicating the S/T-rich motifs in its amino-acid sequence. The ERa point mutants (M1, M2, M3, and M4) were constructed starting from the FH-ERa-WT vector are schematically reported below the wild-type protein. On the right of each schematic protein is summarized its SPOP-binding capacity, sensitivity to SPOP-induced degradation or ubiquitination. (b) The S/T-rich motifs in ERa are required for its binding to SPOP. The 293T cells were transfected with the indicated constructs. After 24 h, cell lysates were prepared for co-IP assay with anti-FLAG antibody and WB analyzes. (c) The S/T-rich motifs in ERa are required for SPOP-mediated ERa degradation. The 293T cells were transfected with the indicated constructs. After 24 h, cell lysates were prepared for WB analyzes. SE, short exposure; LE, long exposure. (d, e) Mutation of the S/T-rich motifs prolongs the half-life of ERa. ERa-WT or M4 mutant was transfected into 293T cells. After 24 h, cells were treated with 30 µM CHX. At the indicated time points, cell lysates were prepared for WB analyzes (d). At each time point, the intensity of ERa was first normalized to the intensity of Actin and then to the value of the 0-h time point (e). (f )The S/T-rich motifs are required for SPOP-mediated ERa polyubiquitination. The 293T cells were transfected with the indicated constructs. After 24 h, cells were treated with 20 µM MG132 for 4 h and the cell lysates were prepared for co-IP assay with anti-FLAG antibody and WB analyzes. The mean values (S.D.) of three independent experiments are shown. (g) Ser118 of ERa is not required for SPOP-mediated ERa degradation. The 293T cells were transfected with FH-ERa or S118A mutant in combination with or without Myc-SPOP constructs. After 24 h, cell lysates were prepared for WB analyzes
Fig 5: PVT1 in exosomes inhibits degradation and ubiquitination of ERG in osteosarcoma cells. Ssos-2 and MNNG/HOS cells were transfected with siRNA of PVT1 (si-PVT1) for 48 h. (A) The expression of ERG protein. (B) The degradation of ERG protein at 3, 6, and 9 hours after the treatment of the protein synthesis inhibitor, CHX (125 µg/mL). (C) The SPOP and ERG proteins were detected in PVT1-protein complex using RNA pull-down assay. Input was used as the positive control; antisense RNA was used as the negative control. (D) PVT1 was detected in ERG-RNA binding complex using RIP assay. Input was used as the positive control; IgG was used as the negative control. (E) Ubiquitination assay: Ssos-2, MG-63, and MNNG/HOS cells were transfected with pcDNA-PVT1, HA-Ub and His-SPOP for 24 h followed by the immunoprecipitation with HA antibody and immunoblotting with ERG antibody. (F) The ubiquitination assay was also performed in PVT1-interfering osteosarcoma cells after being co-cultured with BMSC-EXO. Three independent experiments. *p<0.05, **p<0.01 vs si-control. CHX, cycloheximide. pcDNA-PVT1, the PVT1 overexpressing vector. HA, hemagglutinin. Ub, ubiquitin.
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