(A) Immunopurification and mass spectrometric analysis of MRE11-, NBS1-, or MDC1-containing protein complexes. Cellular extracts from HeLa cells stably expressing FLAG-MRE11, FLAG-NBS1, or FLAG-MDC1 were immunopurified with anti-FLAG affinity beads and eluted with FLAG peptide. The eluates were resolved on SDS/PAGE and silver stained, followed by mass spectrometric analysis. The percentage of peptide coverage of the indicated proteins is shown. (B) Co-IP analysis of the association between USP7 and the MRN-MDC1 complex. Whole-cell lysates from HeLa and MCF-7 cells were immunoprecipitated and then immunoblotted with antibodies against the indicated proteins. α, anti-. (C) FPLC analysis of the native protein complex. Nuclear extracts from HeLa cells were fractionated on Superose 6 size-exclusion columns with high-salt buffer. Chromatographic elution profiles and Western blot analysis of the chromatographic fractions with antibodies against the indicated proteins are shown. Equal volumes from each fraction were analyzed, and the elution positions of the calibration proteins with known molecular masses (kDa) are indicated. The boxed area indicates fractions in which endogenous USP7 was coeluted with the MRN-MDC1 complex. (D) Experiments analogous to those for C were performed with a USP7-containing protein complex purified from FLAG-USP7–expressing HeLa cells. The boxed area indicates fractions in which FLAG-USP7 was coeluted with the MRN-MDC1 complex. (E) Confocal microscopic analysis of USP7 and MRN-MDC1 complex subcellular localization. HeLa cells were fixed and immunostained with antibodies against the indicated proteins. Representative images from biological triplicate experiments are shown. Scale bars: 10 μm.

(A) qChIP analysis of USP7 recruitment around sites of DSBs. HeLa cells stably expressing HA-ER-AsiSI were cultured in the absence or presence of 0.5 μM 4-OHT. qChIP experiments were performed using anti-USP7 or anti-γH2AX antibodies with primers that covered the DNA sequences flanking the AsiSI cutting site and the distal region of the break. γH2AX was used as a positive control. Data represent the mean ± SD of biological triplicate experiments. **P < 0.01, by 2-tailed unpaired Student’s t test. (B) Confocal microscopic analysis of USP7 foci formation upon DSBs. Cells used in A (top) or U2OS cells under x-ray–induced IR (10 Gy, bottom) were preextracted with CSK buffer and then fixed and immunostained with antibodies against USP7 and MDC1 or γH2AX. Scale bars: 10 μm. (C) HeLa cells stably expressing GFP-USP7 were subjected to laser micro-IR and live-cell imaging at the indicated time points. Scale bar: 10 μm. (D) HeLa cells subjected to UVA laser microdissection were fixed and immunostained with antibodies against USP7 and γH2AX or USP7 and MDC1, followed by confocal microscopic analysis. Images are higher-magnification cropped versions of those shown in . Scale bars: 10 μm. (E) HeLa cells were transfected with the indicated siRNAs and subjected to UVA laser microdissection. Then, cells were fixed and immunostained with antibodies against γH2AX or USP7, followed by confocal microscopic analysis. Images are higher-magnification cropped versions of those shown in . Scale bars: 10 μm. (F) HeLa cells stably expressing GFP-USP7 were transfected with the indicated siRNAs, and cells were then subjected to laser micro-IR and live-cell imaging. Scale bars: 10 μm. (B–F) Representative images from biological triplicate experiments are shown.

(A) MCF-7 cells transfected with control siRNA or different sets of USP7 siRNAs were collected and analyzed by Western blotting and qRT-PCR, respectively. (B) Experiments analogous to the data in A were performed using HeLa cells. (C) Cellular lysates or total RNA from WT and USP7-KO HeLa cells were analyzed by Western blotting and qRT-PCR, respectively. (D) USP7-KO HeLa cells were transfected with control vector, WT USP7 (USP7/WT), or UBL domain–deficient USP7 (USP7/ΔUBL) cells. Cellular extracts were prepared and analyzed by Western blotting. (E) MCF-7 cells were transfected with control siRNA or USP7 siRNA, followed by treatment with CHX (50 μg/ml), and harvested at the indicated time points, followed by Western blotting. (F) MCF-7 cells (left) or U2OS cells (right) transfected with control siRNA or USP7 siRNAs were treated with DMSO or the proteasome inhibitor MG132 (10 μM). Cellular extracts were prepared and analyzed by Western blotting. (G) HeLa cells stably expressing FLAG-MDC1 were transfected with control siRNA or NBS1 siRNAs, and whole-cell lysates were collected for co-IP analysis. (H) HeLa cells transfected with control siRNA or different sets of NBS1 siRNAs were collected and analyzed by Western blotting and qRT-PCR, respectively. (I) USP7-KO HeLa cells were transfected with control vector, USP7/WT, or MATH domain–deficient USP7 (USP7/ΔMATH). Cellular extracts were prepared and analyzed by Western blotting. (J) HeLa cells stably expressing GFP-MDC1 were transfected with FLAG-tagged USP7/WT, USP7/ΔUBL, or USP7/ΔMATH. Whole-cell lysates were collected for co-IP analysis. In A–C and H, data represent the mean ± SD of biological triplicate experiments. **P < 0.01, by 1-way ANOVA (A, B and H) and 2-tailed unpaired Student’s t test (C).

(A) HeLa cells with Dox-inducible expression of FLAG-USP7/WT or FLAG-USP7/C223S were transfected with control siRNA or different sets of USP7 5′-UTR siRNAs in the absence or presence of Dox. Cellular extracts were collected and analyzed by Western blotting. (B) HeLa cells were cultured in the absence or presence of increasing amounts of HBX 41,108 for 2 hours as indicated. Cellular extracts and total RNA were collected for Western blot and qRT-PCR analysis, respectively. Data represent the mean ± SD of biological triplicate experiments. P values were calculated by 1-way ANOVA. (C) HeLa cells stably expressing FLAG-MDC1 were cotransfected with control siRNA or USP7 siRNAs and HA-Ub/WT as indicated. Cellular extracts were immunoprecipitated with anti-FLAG followed by IB with anti-HA antibody. (D) HeLa cells stably expressing FLAG-MDC1 were transfected with HA-Ub/WT and cultured in the presence or absence of HBX 41,108. Cellular extracts were immunoprecipitated with anti-FLAG followed by IB with anti-HA antibody. (E) HeLa cells stably expressing FLAG-MDC1 were cotransfected with HA-Ub/WT or HA-Ub/mt and different amounts of Myc-USP7/WT. Cellular extracts were immunoprecipitated with anti-FLAG followed by IB with anti-HA antibody. (F) HeLa cells stably expressing FLAG-MDC1 were cotransfected with HA-Ub/WT and Myc-USP7/WT or different amounts of Myc-USP7/C223S as indicated. Cellular extracts were immunoprecipitated with anti-FLAG antibody followed by IB with anti-HA antibody. (G) HeLa cells stably expressing GFP-MDC1 were cotransfected with HA-Ub/WT and FLAG-USP7/WT, FLAG-USP7/ΔMATH, or FLAG-USP7/ΔUBL. Cellular extracts were immunoprecipitated with anti-GFP antibody followed by IB with anti-HA antibody. (H) HeLa cells stably expressing FLAG-MDC1 were cotransfected with different amounts of Myc-USP7/WT and HA-Ub/K48-only or HA-Ub/K63 only followed by IP analysis. (I) In vitro deubiquitination assays were performed with HA-Ub–conjugated MDC1 purified from HeLa cells using high-salt and detergent buffer and USP7/WT or USP7/C223S purified from baculovirus-infected insect cells.

(A) HeLa cells exposed to IR (6 Gy) or NCS (0.5 μg/ml) were collected at the indicated time points and analyzed by Western blotting with γH2AX as a positive control. (B) Total RNA was collected from HeLa cells exposed to IR or NCS followed by qRT-PCR analysis of MDC1 expression. Data represent the mean ± SD of biological triplicate experiments. P values were calculated by 1-way ANOVA. (C) HeLa cells transfected with control siRNA or USP7 siRNA were exposed to IR and collected at the indicated time points. Expression of the indicated proteins was assessed by Western blotting. (D) HeLa cells or MCF-7 cells were exposed to IR. One hour after IR, cellular extracts were immunoprecipitated and then immunoblotted with antibodies against the indicated proteins. (E) HeLa cells were exposed to UVA (800 kJ/m²) or cultured in the alkylating agent MMC (0.5 μg/ml, 2 h). Cellular extracts were immunoprecipitated and then immunoblotted with antibodies against the indicated proteins. (F) HeLa cells stably expressing FLAG-MDC1 were cotransfected with HA-Ub/WT and USP7 siRNA or Myc-USP7 followed by IR treatment. Cellular extracts were immunoprecipitated with anti-FLAG antibody and then immunoblotted with anti-HA antibody.

(A) HeLa cells stably expressing HA-ER-AsiSI were transfected with USP7 siRNA and cultured in the presence of 0.5 μM 4-OHT for 4 hours. Then, cells were fixed and immunostained with the indicated antibodies followed by confocal microscopic analysis. White arrows indicate cells with different amounts of USP7 depletion. Representative images from biological triplicate experiments are shown. Scale bars: 10 μm. (B) HeLa cells stably expressing control vector or FLAG-MDC1 were cotransfected with the indicated siRNAs and GFP-MRE11. Then, cells were subjected to UV laser micro-IR and live-cell imaging at the indicated time points. Representative images from biological triplicate experiments are shown. Scale bars: 10 μm. (C) Images shown are analogous to those in B, but for GFP-RAD50. Scale bars: 10 μm. (D) Images shown are analogous to those B, but for GFP-NBS1. Scale bars: 10 μm. (E) HeLa cells stably expressing HA-ER-AsiSI were transfected with the indicated siRNAs and control vector, FLAG-MDC1, or Myc-RNF168. Cells were then treated with 0.5 μM 4-OHT, fixed, and immunostained with antibodies against BRCA1 and USP7 followed by confocal microscopic analysis. Representative images from biological triplicate experiments are shown. Scale bars: 10 μm. (F) Images shown are analogous to those in E, but for 53BP1 and USP7. Scale bars: 10 μm. (G) Cellular extracts were collected from cells used in E and F, and expression of the indicated proteins was assessed by Western blotting.

(A) Human tissues containing cervical carcinoma with different grades and tumor-adjacent cervical samples were analyzed by IHC staining. Representative images (original magnification, ×200) are shown. Scale bars: 50 μm. (B) Values for the stainings from A were determined by ImagePro Plus software and are presented with box plots. Boxes represent the 25th and 75th percentiles, lines represent the median, and whiskers show the minimum and maximum points. *P < 0.05 and **P < 0.01, by 1-way ANOVA. The correlation coefficient and P values were analyzed as indicated. (C) Analysis of Pyeon cervix (left) or Biewenga cervix (right) from Oncomine for the expression of USP7 in normal human cervical tissues and cervical carcinoma samples. Data are presented with box plots (**P < 0.01, by unpaired, 2-tailed Student’s t test). (D) Bioinformatics analysis of TCGA data set (downloaded from https://portal.gdc.cancer.gov/projects/TCGA-CESC) for the gene copy numbers of USP7 in cervical paracancerous tissue and cervical carcinoma samples. Data are presented with box plots (**P < 0.01, by unpaired, 2-tailed Student’s t test). (E) Kaplan-Meier survival analysis for the relationship between the survival cervical cancer patients and expression levels of USP7 mRNA, with survival packages from TCFA (https://tcga-data.nci.nih.gov/docs/publications/tcga/). (F) Kaplan-Meier survival analysis of the relationship between the survival of cervical cancer patients and gene copy number status of USP7, with survival packages from TCGA (https://tcga-data.nci.nih.gov/docs/publications/tcga/).

(A) HeLa cells stably expressing the indicated shRNAs and genes were irradiated with different doses of x-ray IR. Colony formation assays were conducted, and representative images from biological triplicate experiments are shown. (B) Experiments analogous to those for A were performed using SiHa cells. Representative images from biological triplicate experiments are shown. (C) HeLa cells stably expressing the indicated shRNAs and GFP vector or GFP-MDC1 were cocultured with equivalent native HeLa cells. The mixed cells were then irradiated with different doses of x-ray IR and analyzed by FACS 10 days later. The percentage of GFP-positive cells relative to GFP-negative cells under IR treatment was normalized to percentages in the untreated control mixture. The ratio reflects cellular fitness. Data represent the mean ± SD from biological triplicate experiments. *P < 0.05 and **P < 0.01, by 1-way ANOVA. (D) Experiments analogous to those for C were performed using SiHa cells. Data represent the mean ± SD from biological triplicate experiments. *P < 0.05 and **P < 0.01, by 1-way ANOVA. (E) Cell viability analysis was performed with SiHa cells stably expressing vector or FLAG-MDC1. Cells were cultured in increasing amounts of GNE-6640 or GNE-6776 followed by x-ray IR (2 Gy). Data represent the mean ± SD from biological triplicate experiments. **P < 0.01, by 2-way ANOVA. (F) SiHa tumors stably expressing shRNAs and the indicated genes were transplanted into athymic mice (n = 12), and half of the mice in each group were randomly chosen and subjected to 10 Gy x-ray IR 1 week after tumor transplantation. Tumor volumes were measured weekly, and tumors were harvested and weighed when mice were sacrificed. Data represent the mean ± SD. *P < 0.05 and **P < 0.01, by 2-way ANOVA for tumor volume analysis and 1-way ANOVA for tumor weight analysis. Images of representative tumors and sacrificed mice are shown.