Journal: Molecular carcinogenesis

Article Title: USP9X deubiquitinates and stabilizes CDC123 to promote breast carcinogenesis through regulating cell cycle.

doi: 10.1002/mc.23591

Figure Lengend Snippet: FIGURE 1 Cell division cycle 123 (CDC123) deregulation contributes to breast carcinogenesis. (A) Expression of CDC123 in normal and tumor samples from different tissues analyzed in the GEPIA2 database. Bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), cholangio carcinoma (CHOL), colon adenocarcinoma (COAD), lymphoid neoplasm diffuse large B‐cell lymphoma (DLBC), glioblastoma multiforme (GBM), brain lower grade glioma (LGG), liver hepatocellular carcinoma (LIHC), lung squamous cell carcinoma (LUSC), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), rectum adenocarcinoma (READ), sarcoma (SARC), stomach adenocarcinoma (STAD), testicular germ cell tumors (TGCT), thymoma (THYM), uterine corpus endometrial carcinoma (UCEC), uterine carcinosarcoma (UCS). (B) Box plot of CDC123 transcription levels in different grades of breast cancer analyzed in the GEPIA2 database. (C) Correlation analysis between CDC123 expression level and adverse overall survival. The p value was determined by one‐way analysis of variance (ANOVA). (D) Colony formation assays were conducted with MCF‐7 cells stably expressing the shRNA targeting CDC123. Representative images from biological triplicate experiments are shown (left panel). The colony numbers were statisticed (right panel). (E) Soft agar assays to assess anchorage‐independent growth were conducted with MCF‐7 cells stably expressing the indicated shRNAs. Representative images from biological triplicate experiments are shown (left panel). The number of diameter > 20 um colonies were statisticed (right panel). Scale bar = 100 μm. (F, G) Control or CDC123‐deficient MCF‐7 tumors were transplanted into athymic mice, and tumors were harvested 8 weeks later, tumor volume was shown (G). (H) The tumor weight was shown (left panel), the expression of CDC123 was examined by western blot analysis (right panel). Each bar represents the mean ± standard deviation for tumor weight measurements (n = 5, in each group). **p < 0.01, one‐way ANOVA.

Article Snippet: The antibodies used in the article are shown below: CDC123 (SC‐ 365596) (1:1000 for WB), HA (sc‐805) (1:2000 for WB) from Santa Cruz Biotechnology; USP9X (55054‐1‐AP) (1:1000 for WB) from Proteintech; β‐actin (A1978) (1:1000 for WB), FLAG (F3165) (1:2000 for WB; 1:500 for IP) from Sigma; Myc (M047‐3) (1:1000 for WB) from MBL.

Techniques: Expressing, Stable Transfection, shRNA, Control, Western Blot, Standard Deviation

Journal: Molecular carcinogenesis

Article Title: USP9X deubiquitinates and stabilizes CDC123 to promote breast carcinogenesis through regulating cell cycle.

doi: 10.1002/mc.23591

Figure Lengend Snippet: FIGURE 2 Deubiquitinase ubiquitin‐specific peptidase 9, X‐linked (USP9X) is physically associated with cell division cycle 123 (CDC123). (A) Immunoaffinity purification of CDC123‐containing protein complexes. Whole‐cell extracts from MCF‐7 cells with expression of stably integrated FLAG‐CDC123 were purified with an anti‐FLAG affinity column. After extensive washing, the bound proteins were eluted with excess FLAG peptides, resolved, and visualized by silver staining on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gels. The protein bands on the gel were recovered and analyzed by mass spectrometry. Representative peptide fragments of USP9X and CDC123 and peptide coverage of the indicated proteins are shown. Detailed results from the mass spectrometric analysis are provided in Supporting Information File 1. (B) Co‐immunoprecipitation analysis of the association between CDC123 and USP9X. Whole‐cell lysates from MCF‐7 cells and 231 cells were immunoprecipitated (IP) and then immunoblotted (IB) with antibodies against the indicated proteins. (C) Immunostaining and confocal microscopy analysis of CDC123 and USP9X subcellular localization. Scale bar = 20 μm. (D) Co‐immunoprecipitation analysis of the association between CDC123 and USP9X. FLAG‐tagged deletion mutants of USP9X were transfected into MCF‐7 cells followed by co‐immunoprecipitation analysis. The conserved domains of USP9X were determined by the SMART program.

Article Snippet: The antibodies used in the article are shown below: CDC123 (SC‐ 365596) (1:1000 for WB), HA (sc‐805) (1:2000 for WB) from Santa Cruz Biotechnology; USP9X (55054‐1‐AP) (1:1000 for WB) from Proteintech; β‐actin (A1978) (1:1000 for WB), FLAG (F3165) (1:2000 for WB; 1:500 for IP) from Sigma; Myc (M047‐3) (1:1000 for WB) from MBL.

Techniques: Ubiquitin Proteomics, Immunoaffinity Purification, Expressing, Stable Transfection, Purification, Affinity Column, Silver Staining, Polyacrylamide Gel Electrophoresis, Mass Spectrometry, Immunoprecipitation, Immunostaining, Confocal Microscopy, Transfection

Journal: Molecular carcinogenesis

Article Title: USP9X deubiquitinates and stabilizes CDC123 to promote breast carcinogenesis through regulating cell cycle.

doi: 10.1002/mc.23591

Figure Lengend Snippet: FIGURE 3 Ubiquitin‐specific peptidase 9, X‐linked (USP9X) promotes cell division cycle 123 (CDC123) stabilization. (A) MCF‐7 cells were transfected with control siRNA or different sets of USP9X siRNAs. Cellular extracts and total RNA were prepared and analyzed by western blot analysis and quantitative real‐time reverse‐transcription polymerase chain reaction (qRT‐PCR), respectively. Each bar represents the mean ± standard deviation (SD) for biological triplicate experiments. **p < 0.01, one‐way analysis of variance (ANOVA). (B) MCF‐7 cells were transfected with control siRNA or USP9X siRNA followed by treatment with DMSO or proteasome inhibitor MG132 (10 μM). Cellular extracts were prepared and analyzed by Western blot analysis. (C) MCF‐7 cells transfected with control siRNA or USP9X siRNA were treated with cycloheximide (CHX) and harvested at the indicated time followed by western blot analysis analysis. (D) MCF‐7 cells with Dox‐inducible expression of FLAG‐USP9X/wt or FLAG‐USP9X/C1566S were cultured in the absence or presence of increasing amounts of Dox. Cellular extracts were collected and analyzed by western blot analysis and qRT‐PCR, respectively. Each bar represents the mean ± SD for biological triplicate experiments, **p < 0.01, one way ANOVA. (E) MCF‐7 cells were cultured in the absence or presence of WP1130 for 24 h as indicated concentration. Cellular extracts and total RNA were collected and analyzed by western blot analysis and qRT‐PCR, respectively. Each bar represents the mean ± SD for biological triplicate experiments. p Values were determined by one‐way ANOVA.

Article Snippet: The antibodies used in the article are shown below: CDC123 (SC‐ 365596) (1:1000 for WB), HA (sc‐805) (1:2000 for WB) from Santa Cruz Biotechnology; USP9X (55054‐1‐AP) (1:1000 for WB) from Proteintech; β‐actin (A1978) (1:1000 for WB), FLAG (F3165) (1:2000 for WB; 1:500 for IP) from Sigma; Myc (M047‐3) (1:1000 for WB) from MBL.

Techniques: Ubiquitin Proteomics, Transfection, Control, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation, Expressing, Cell Culture, Concentration Assay

Journal: Molecular carcinogenesis

Article Title: USP9X deubiquitinates and stabilizes CDC123 to promote breast carcinogenesis through regulating cell cycle.

doi: 10.1002/mc.23591

Figure Lengend Snippet: FIGURE 4 Ubiquitin‐specific peptidase 9, X‐linked (USP9X) promotes cell division cycle 123 (CDC123) deubiquitination. (A) Stable isotope labeling with amino acids in cell culture (SILAC)‐based quantitative mass spectrometry analysis of possible substrate proteins for USP9X with MCF‐7 cells. Control cells were labeled with heavy isotopic lysine and arginine (K8R10) and shUSP9X‐treated cells were labeled with light isotopic lysine and arginine (K0R0). Cellular extracts were desalted by gel separation and mixed for digestion followed by mass spectrometry analysis. (B) Gene ontology (GO) enrichment analysis was performed on the proteins modified with ubiquitination after USP9X knockdown. (C) Volcano map showing proteins from SILAC of USP9X knockdown in MCF‐7 cells. (D) MCF‐7 cells (left panel) and 231 cells (right panel) stably expressing FLAG‐CDC123 were co‐transfected with control siRNA or USP9X siRNAs together with HA‐Ub/wt as indicated. Cellular extracts were prepared for co‐immunoprecipitation assays with anti‐FLAG followed by IB with anti‐HA. (E) MCF‐7 cells expressing FLAG‐USP9X/wt or FLAG‐USP9X/C1566S were co‐transfected with Myc‐CDC123 and HA‐Ub/wt. Cellular extracts were prepared for co‐immunoprecipitation assays with anti‐Myc followed by IB with anti‐HA. (F) MCF‐7 cells stably expressing Myc‐CDC123 were cultured in the presence of different concentrations of WP1130. Cellular extracts were prepared for co‐immunoprecipitation assays with anti‐Myc followed by IB with anti‐HA. (G) MCF‐7 cells stably expressing Myc‐CDC123 were co‐transfected with FLAG‐USP9X and HA‐Ub/wt, HA‐Ub/K63‐only or HA‐Ub/K48‐only. Cellular extracts were prepared for co‐immunoprecipitation assays with anti‐Myc followed by IB with anti‐HA. (H) In vitro deubiquitination assays with MCF‐7 cells‐purified Myc‐tagged CDC123/wt‐HA‐Ub or CDC123/K308R‐HA‐Ub and FLAG‐USP9X/wt. (I) Co‐immunoprecipitation of the association of USP9X with FLAG‐CDC123/wt or FLAG‐CDC123/K308R.

Article Snippet: The antibodies used in the article are shown below: CDC123 (SC‐ 365596) (1:1000 for WB), HA (sc‐805) (1:2000 for WB) from Santa Cruz Biotechnology; USP9X (55054‐1‐AP) (1:1000 for WB) from Proteintech; β‐actin (A1978) (1:1000 for WB), FLAG (F3165) (1:2000 for WB; 1:500 for IP) from Sigma; Myc (M047‐3) (1:1000 for WB) from MBL.

Techniques: Ubiquitin Proteomics, Quantitative Proteomics, Cell Culture, Multiplex sample analysis, Mass Spectrometry, Control, Labeling, Modification, Knockdown, Stable Transfection, Expressing, Transfection, Immunoprecipitation, In Vitro, Purification

Journal: Molecular carcinogenesis

Article Title: USP9X deubiquitinates and stabilizes CDC123 to promote breast carcinogenesis through regulating cell cycle.

doi: 10.1002/mc.23591

Figure Lengend Snippet: FIGURE 5 Ubiquitin‐specific peptidase 9, X‐linked (USP9X)/cell division cycle 123 (CDC123) axis regulates the cell cycle. (A) Heatmap of DEGs in CDC123‐siRNA‐treated and USP9X‐siRNA‐treated MCF‐7 cells normalized with control‐siRNA‐treated in MCF‐7 cells based on RNA‐ seq (left panel). The numbers of overlapping, downregulated DEGs in CDC123 and USP9X depleted MCF‐7 cells using three different targeting siRNAs (right panel). (B) GSEA of 792 downregulated overlapped genes with −log10 plot of the uncorrected P value on the x‐axis. (C) MCF‐7 cells were transfected with the indicated siRNAs followed by RNA extraction and quantitative real‐time reverse‐transcription polymerase chain reaction analysis of the expression of the indicated genes. (D, E) Control cells or MCF‐7 cells were transfected with the indicated siRNAs and cell cycle profiles were analyzed by fluorescence‐activated cell sorting, cell populations were shown (E). (F) Cellular lysates from these cells were analyzed by western blot analysis with antibodies against the indicated proteins. Each bar represents the mean ± standard deviation for biological triplicate experiments. *p < 0.05; **p < 0.01, one‐way analysis of variance.

Article Snippet: The antibodies used in the article are shown below: CDC123 (SC‐ 365596) (1:1000 for WB), HA (sc‐805) (1:2000 for WB) from Santa Cruz Biotechnology; USP9X (55054‐1‐AP) (1:1000 for WB) from Proteintech; β‐actin (A1978) (1:1000 for WB), FLAG (F3165) (1:2000 for WB; 1:500 for IP) from Sigma; Myc (M047‐3) (1:1000 for WB) from MBL.

Techniques: Ubiquitin Proteomics, Control, RNA Sequencing, Transfection, RNA Extraction, Reverse Transcription, Polymerase Chain Reaction, Expressing, Fluorescence, FACS, Western Blot, Standard Deviation

Journal: Molecular carcinogenesis

Article Title: USP9X deubiquitinates and stabilizes CDC123 to promote breast carcinogenesis through regulating cell cycle.

doi: 10.1002/mc.23591

Figure Lengend Snippet: FIGURE 7 Ubiquitin‐specific peptidase 9, X‐linked (USP9X)/cell division cycle 123 (CDC123) promotes breast carcinogenesis through regulating cell cycle. Mechanistic model of USP9X/CDC123 promoting breast carcinogenesis through cell cycle regulation. In normal, USP9X interacts with CDC123 and deubiquitinate K48‐linked ubiquitinated CDC123 at the K308 site. This process enhances the stability of CDC123 and maintains the expression of cell cycle‐related genes, thereby promoting breast cancer cell proliferation and invasion. However, disruption of USP9X expression or treatment with the USP9X deubiquitinase inhibitor WP1130 leads to the accumulation of K48‐linked ubiquitinated CDC123 and subsequent degradation of CDC123. As a result, the expression of cell cycle‐related genes such as CCNB1, CCNA2, FOXM1 is downregulated. Thereby, the cell population in the G0/G1 phase is accumulated and results in breast cancer cell proliferation and invasion inhibition. In summary, our study identified USP9X as a key player in deubiquitinating and stabilizing CDC123, thereby promoting breast carcinogenesis through regulation of the cell cycle.

Article Snippet: The antibodies used in the article are shown below: CDC123 (SC‐ 365596) (1:1000 for WB), HA (sc‐805) (1:2000 for WB) from Santa Cruz Biotechnology; USP9X (55054‐1‐AP) (1:1000 for WB) from Proteintech; β‐actin (A1978) (1:1000 for WB), FLAG (F3165) (1:2000 for WB; 1:500 for IP) from Sigma; Myc (M047‐3) (1:1000 for WB) from MBL.

Techniques: Ubiquitin Proteomics, Expressing, Disruption, Inhibition