Fig 2: Knockdown USP19 induces the formation of anaphase bridgeA. Selected frames from time-lapse movies of representative HeLa/GFP-H2B cells transfected with control siRNA (siCtrl) or USP19 siRNA (siUSP19). Arrows denote segregation error. The time on the images is in minutes. NEB, nuclear envelope breakdown; M, metaphase; A, anaphase. Scale bar, 5 μm. B. The percentage of segregation errors in control and USP19 knockdown cells during anaphase. Data are representative of three independent experiments, error bars indicate S.D. C. Immunoblot analyzes knockdown efficiency of USP19. D. HCT116 cells were transfected with control or USP19 siRNA, then synchronized in mitosis by thymidine block and release. The cells through mitosis were immunostained with anti-centromere antibodies (ACA; for kinetochores; red) and DAPI (for chromosomes; blue). Upper panels shows example images of normal segregation, anaphase bridges and lagging chromosomes in anaphase. Scale bar, 5 μm. Lower panels is quantification of mitotic cells with anaphase bridges or lagging chromosomes in control and USP19 knockdown cells. *p<0.05, **p<0.01. E, F. Complementation of RFP-USP19 in knockdown HeLa/GFP-H2B cells rescues chromosome segregation errors. The USP19 knockdown cells were transfected with USP19 siRNA-resistant expression construct or RFP vector. (E) The expression of siRNA-resistant RFP-USP19 was detected by Immunoblot. (F) The data show the percentage of mitotic cells with segregation errors during anaphase in RFP-positive cells, data are shown as mean ± S.D. *p<0.05, **p<0.01.

Fig 3: USP19 expression in human cancers and schematic presentation roles of USP19 in DNA damage responseA. Copy number status of USP19 in various types of human tumor samples from the database of cBioportal for cancer genomics ( http://www.cbioportal.org/index.do. Data source which contains more than 150 clinical samples that are mostly based on TCGA were analyzed, including kidney renal clear cell carcinoma (TCGA provisional), stomach adenocarcinoma (TCGA Nature, 2014), cervical squamous cell carcinoma (TCGA provisional), esophageal carcinoma (TCGA provisional) and brain lower grade glioma (TCGA provisional). B. The tissue array of kidney renal clear cell carcinoma was performed by immunohistochemistry with anti-USP19 antibody. USP19 expression was plotted using the score as described in the ‘Materials and Methods’ section. Outliers are indicated by open circles, extremes by asterisks. C. Representative images from immunohistochemical staining of USP19 in adjacent normal tissue and kidney renal clear cell carcinoma; scale bar, 50μm. D. Schematic presentation roles of USP19 in DNA damage response. In response to IR irradiation, USP19 translocate into nucleus and physically interacts with HDAC1/2 for regulating their K63-linked ubiquitination and involves in NHEJ mediated DNA repair. Due to the defect in DNA repair, cells with USP19 depletion showed accumulated DNA damage which would lead to the formation of chromosome error and finally contribute to genomic instability and even tumorigenesis.

Fig 4: USP19 specifically deubiquitinates HDAC1/2 for their K63-linked ubiquitinationA. HEK293T cells were transfected with Myc-tagged Ubiquitin (Myc-Ub), Flag-HDAC1, Flag-HDAC2, HA-USP19 WT, HA-USP19 CA as indicated. Cell lysates were extracted under denaturing conditions (95°C, 1% SDS) and immunoprecipitated with anti-Flag, immune complexes were immunoblotted with anti-Flag. B. Seventy-two hours after Control or USP19 siRNA transfection, protein levels of HDAC1, HDAC2 and USP19 were assessed by Immunoblot. C. HA-USP19 WT, HA-USP19 CA or HA vector was transfected into cells. Seventy-two hours after transfection, protein levels of HDAC1, HDAC2 and HA-USP19 were assessed by Immunoblot. D. 293T cells were transfected with Myc-Ub, Flag-HDAC1, Flag-HDAC2, HA-USP19 WT, and HA-USP19 CA as indicated. Cell lysates were extracted under denaturing conditions (95°C, 1% SDS), then immunoprecipitated with anti-Flag and immunoblotted with linkage-specific antibodies recognizing K63-linked polyubiquitin.

Fig 5: USP19 is required for in NHEJ-mediated DNA repairA. EJ5-GFP U2OS cells were cotransfected with control or USP19 siRNA and HA-I-SceI. Cells were collected and analyzed for the proportion of GFP-positive cells by flow cytometric. The NHEJ efficiency presented as relative quantification compared with control siRNA. Data are shown as mean ± S.D. *p<0.05, **p<0.01. B. DR-GFP U2OS cells were cotransfected with control or USP19 siRNA and HA-I-SceI. Cells were collected and analyzed for the proportion of GFP-positive cells by flow cytometric. The HR efficiency presented as relative quantification compared with control siRNA. Data are shown as mean ± S.D. C. EJ5-GFP U2OS cells were cotransfected with HA-I-SceI and siRNAs as indicated. Protein levels of HDAC1, HDAC2, USP19 and HA-I-SceI were assessed by Immunoblot. D. EJ5-GFP U2OS cells were cotransfected with HA-I-SceI and siRNAs as in C. Cells were collected and analyzed for the proportion of GFP-positive cells by flow cytometric. The NHEJ efficiency presented as relative quantification compared with control siRNA. Data are shown as mean ± S.D. *p<0.05, **p<0.01.