Journal: NPJ Breast Cancer

Article Title: Inhibition of CXorf56 promotes PARP inhibitor-induced cytotoxicity in triple-negative breast cancer

doi: 10.1038/s41523-023-00540-3

Figure Lengend Snippet: A The schematic diagram highlights the criteria for gene selection and the experimental setup for the RNAi screen. #1 890 DDR-related genes were identified based on the CRISPR-Cas9 screens against 27 genotoxic agents . #2 The top 500 overall-survival-related genes were directly downloaded through GEPIA portal based on the TCGA-BRCA database . B The heat map that represents the expression of selected genes in different BC cells was determined by qPCR analysis. C The bar chart shows the average Z-score of TNBC cell lines minus the average Z-score of luminal cell lines (ΔZ-score) for all the genes included in the RNAi screen. A negative value indicates that the siRNA decreased the HR/NHEJ ratio more in the TNBC cell lines (* P < 0.05 comparing TNBC and luminal Z-scores). Data were analyzed by a two-tailed t test. D The average TNBC (T) and luminal (L) Z-scores for all cell lines for CXorf56 . The P -value (**** P < 0.0001) indicates a statistically significant difference between TNBC and luminal lines. Data were analyzed by a two-tailed t test. E The difference in CXorf56 transcript levels between normal breast and cancer tissues is presented as a scatter dot plot. Data were analyzed by a two-tailed t test. Scatter dot plots show the transcript levels of CXorf56 across the breast cancer subtypes based on the IHC ( F ) or PAM50 ( G ) classification of the TCGA-BRCA cohort. Data were analyzed by a two-tailed t test. H Kaplan-Meier survival curve shows a poorer OS rate in BC patients with high CXorf56 transcript levels than those with low CXorf56 transcript levels. Data were analyzed by a log-rank test. Remarks: * p < 0.05, ** p < 0.01, *** p < 0.001, **** P < 0.0001; Data are presented as mean ± SD.

Article Snippet: Supernatants were incubated overnight at 4 °C with CXorf56 protein antibody (catalog number 24021-1-AP, Proteintech, Rosemont, IL, USA), followed by immunoprecipitation (6 h, 4 °C) with agarose beads (Amersham Biosciences, Amersham, UK).

Techniques: Selection, CRISPR, Expressing, Two Tailed Test

Journal: NPJ Breast Cancer

Article Title: Inhibition of CXorf56 promotes PARP inhibitor-induced cytotoxicity in triple-negative breast cancer

doi: 10.1038/s41523-023-00540-3

Figure Lengend Snippet: A Representative IHC images of CXorf56 protein expression (nuclear staining pattern) in non-TNBC tissues and TNBC tissues; Scale bar = 50 μm. The scores indicate CXorf56 protein levels in tumor tissues in different subgroups ( B , non-TNBC vs. TNBC; C , stage I-II vs. stage III; D , tumor size <3 cm vs. tumor size >3 cm). The scores were calculated by the intensity and percentage of stained cells as described in the methods. Data were analyzed by a two-tailed t test. E BC patients with high CXorf56 protein expression (score of 4–7) have poorer OS than patients with low CXorf56 protein expression (score of 0–3). Data were analyzed by a log-rank test. F The expression of CXorf56 protein is an independent prognostic factor for OS of BC patients, based on the multivariate Cox proportional hazard model, which is shown in the forest plot. Data were analyzed by a Wald test. Remarks: ns p ≥ 0.05, *** p < 0.001, **** P < 0.0001.

Article Snippet: Supernatants were incubated overnight at 4 °C with CXorf56 protein antibody (catalog number 24021-1-AP, Proteintech, Rosemont, IL, USA), followed by immunoprecipitation (6 h, 4 °C) with agarose beads (Amersham Biosciences, Amersham, UK).

Techniques: Expressing, Staining, Two Tailed Test

Journal: NPJ Breast Cancer

Article Title: Inhibition of CXorf56 promotes PARP inhibitor-induced cytotoxicity in triple-negative breast cancer

doi: 10.1038/s41523-023-00540-3

Figure Lengend Snippet: CXorf56 stable knockdown efficiency was detected by qPCR in TNBC cells including MDA-MB-231 ( A ), BT549 ( B ), and SUM1315 ( C ) cells, respectively. Data were analyzed by a two-tailed t test. Negative control (NTC) and CXorf56 -knockdown TNBC cells ( D , E , MDA-MB-231; F , G , BT549; H , I , SUM1315) were treated without (Ctrl) or with IR (2 Gy), γ-H2AX foci before or 8 hours after IR was detected by immunofluorescence. Nuclei were visualized with DAPI (blue). Representative images of γ-H2AX foci are shown in D , F , H . Quantification of focus signals is shown in E , G , I . Data were analyzed by ANOVA and two-tailed t test. Remarks: * p < 0.05, ** p < 0.01, *** p < 0.001; Data are presented as mean ± SD. Scale bars = 10 μm.

Article Snippet: Supernatants were incubated overnight at 4 °C with CXorf56 protein antibody (catalog number 24021-1-AP, Proteintech, Rosemont, IL, USA), followed by immunoprecipitation (6 h, 4 °C) with agarose beads (Amersham Biosciences, Amersham, UK).

Techniques: Knockdown, Two Tailed Test, Negative Control, Immunofluorescence

Journal: NPJ Breast Cancer

Article Title: Inhibition of CXorf56 promotes PARP inhibitor-induced cytotoxicity in triple-negative breast cancer

doi: 10.1038/s41523-023-00540-3

Figure Lengend Snippet: A Confirmation of CXorf56 protein knockdown (shCX#1 and shCX#2) and re-expression (rescued#1 and rescued#2) in MDA-MB-231 cells. The sensitivity of control (Ctrl), Cxorf56 -knockdown, or CXorf56 re-expression MDA-MB-231 cells to IR ( B ), Cisplatin ( C ), and Olaparib ( D ) was assessed by colony formation assays. Data were analyzed by a two-tailed t test. E , F Ctrl, CXorf56 -knockdown, or CXorf56 re-expression MDA-MB-231 cells, were transfected with NHEJ reporter (EJ5-GFP) or HR reporter (DR-GFP) along with pCBA-I-SceI and mCherry. Forty-eight hours later, cells were harvested and subjected to flow cytometric analysis. Data were analyzed by a two-tailed t test. G Analysis shows linear regressions and Pearson correlations between relative NHEJ and HR efficiency in Ctrl, CXorf56 -knockdown, and CXorf56 re-expression MDA-MB-231 cells. Data were analyzed by a F test. H Cell cycle analyses of Ctrl, CXorf56 -knockdown, or CXorf56 re-expression MDA-MB-231 cells show that the change of CXorf56 protein levels did not alter the cell cycle distribution of MDA-MB-231 cells. Data were analyzed by ANOVA and two-tailed t test. I , J Ctrl, CXorf56 -knockdown, and CXorf56 re-expression MDA-MB-231 cells were treated with IR (1 Gy, 1 hour for MDC1, 53BP1, Ku70; 1 Gy, 3 hours for RPA32; 1 Gy, 5 hours for BRAC2 and RAD51), and indicated foci were detected by immunofluorescence. Representative images are shown in i . Quantification of focus signals is shown in J . Data were analyzed by ANOVA and two-tailed t test. Remarks: ns p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001; Data are presented as mean ± SD. Scale bars = 10 μm.

Article Snippet: Supernatants were incubated overnight at 4 °C with CXorf56 protein antibody (catalog number 24021-1-AP, Proteintech, Rosemont, IL, USA), followed by immunoprecipitation (6 h, 4 °C) with agarose beads (Amersham Biosciences, Amersham, UK).

Techniques: Knockdown, Expressing, Control, Two Tailed Test, Transfection, Immunofluorescence

Journal: NPJ Breast Cancer

Article Title: Inhibition of CXorf56 promotes PARP inhibitor-induced cytotoxicity in triple-negative breast cancer

doi: 10.1038/s41523-023-00540-3

Figure Lengend Snippet: CXorf56 protein ( A ) and Ku70 ( B ) complexes were co-immunoprecipitated with CXorf56 protein and Ku70 antibodies and immunoblotted with the indicated antibodies. C , D MDA-MB-231 cells were infected with indicated lentiviral plasmids and treated with Olaparib. The indicated protein levels ( C ) and the sensitivity to Olaparib were assessed ( D ). Data were analyzed by a two-tailed t test. E , F NTC and corresponding knockdown MDA-MB-231 cells were treated with Olaparib (1.5 μM), and γ-H2AX foci before or 48 hours after Olaparib treatment were detected by immunofluorescence. Representative images of γ-H2AX foci are shown in E ; Scale bars = 10 μm. Quantification of focus signals is shown in F . Data were analyzed by a two-tailed t test. G , J NTC and corresponding knockdown MDA-MB-231 cells were transfected with NHEJ reporter or HR reporter along with pCBA-I-SceI and mCherry. 48 hours later, cells were harvested and subjected to flow cytometric analysis. Data were analyzed by a two-tailed t test. I – K The tumor growth of the indicated MDA-MB-231 cells was examined in xenografts under the treatment of Olaparib ( n = 6). Mouse xenograft tumors ( I ), the elevation of tumor size for 40 days ( J ), and the weights of the xenograft tumors ( K ) were presented. Data were analyzed by a two-tailed t test. L Representative IHC images of CXorf56 protein, Ku70, and γ-H2AX expression in mouse xenograft tumor tissues; Scale bars = 100 μm. M Differences in the γ-H2AX protein levels in xenograft tumor tissues were presented in the violin plot ( n = 6). Data were analyzed by a two-tailed t test.Remarks: ns p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001; Data are presented as mean ± SD.

Article Snippet: Supernatants were incubated overnight at 4 °C with CXorf56 protein antibody (catalog number 24021-1-AP, Proteintech, Rosemont, IL, USA), followed by immunoprecipitation (6 h, 4 °C) with agarose beads (Amersham Biosciences, Amersham, UK).

Techniques: Immunoprecipitation, Infection, Two Tailed Test, Knockdown, Immunofluorescence, Transfection, Expressing

Journal: NPJ Breast Cancer

Article Title: Inhibition of CXorf56 promotes PARP inhibitor-induced cytotoxicity in triple-negative breast cancer

doi: 10.1038/s41523-023-00540-3

Figure Lengend Snippet: A LMNA (lamin A) Cas9 reporter. CRISPR-Cas9 and sgRNA target the 5’ UTR of LMNA. Cas9 with sgRNA generates DSBs. HR fuses mClover with a start codon (red arrow) to LMNA for repair. mClover protein levels ( B ) were investigated by western blotting and the average percentage of mClover+ cells ( C ) in indicated MDA-MB-231 cells in five independent experiments, normalized to the vector control (Vec ctrl) group. Data were analyzed by a two-tailed t test. D , E Corresponding MDA-MB-231 cells were treated with IR (1 Gy, 1 hour for DNA-PKcs, XRCC4, and LIG4), and indicated foci were detected by immunofluorescence. Representative images are shown in D , Scale bars = 10 μm., and quantification of focus signals is shown in E . Data were analyzed by a two-tailed t test. F – H MDA-MB-231 cells were synchronized with nocodazole (100 ng/ml) for 12 hours and released into the cell cycle. At the indicated time points, cells were harvested for cell cycle ( F ) and co-immunoprecipitation and Western blotting analysis without IR treatment ( G ) or with 1-hour of IR treatment (2 Gy) ( H ). I Schematic of the domains of Ku70. There are three main domains in Ku70: the N-terminal vWA domain (blue), the core DNA binding domain (DBD, yellow), and the C-terminal domain (CTD, gray). J MDA-MB-231 cells were transfected with plasmids encoding the indicated Flag-tagged truncated Ku70 constructs. Cell lysates were subjected to immunoprecipitation with an anti- Cxorf56 protein antibody. Precipitated proteins were analyzed by Western blotting with an anti-Flag antibody. Input controls were also included. Remarks: ns p ≥ 0.05, * p < 0.05, ** p < 0.01; Data are presented as mean ± SD.

Article Snippet: Supernatants were incubated overnight at 4 °C with CXorf56 protein antibody (catalog number 24021-1-AP, Proteintech, Rosemont, IL, USA), followed by immunoprecipitation (6 h, 4 °C) with agarose beads (Amersham Biosciences, Amersham, UK).

Techniques: CRISPR, Western Blot, Plasmid Preparation, Control, Two Tailed Test, Immunofluorescence, Immunoprecipitation, Binding Assay, Transfection, Construct

Journal: NPJ Breast Cancer

Article Title: Inhibition of CXorf56 promotes PARP inhibitor-induced cytotoxicity in triple-negative breast cancer

doi: 10.1038/s41523-023-00540-3

Figure Lengend Snippet: A Upregulated CXorf56 protein naturally binds to Ku70, which blocks the binding of Ku70 with the damaged DNA, inducing a selection of HR over NHEJ by competitive recruitment of HR regulators for the repair of DSBs. B Without the binding of CXorf56 protein with Ku70, Ku70 is recruited into chromatin at DSB sites and activates the NHEJ pathway by further forming the phospho-DNA-PKcs, XRCC4, and LIG4 complex.

Article Snippet: Supernatants were incubated overnight at 4 °C with CXorf56 protein antibody (catalog number 24021-1-AP, Proteintech, Rosemont, IL, USA), followed by immunoprecipitation (6 h, 4 °C) with agarose beads (Amersham Biosciences, Amersham, UK).

Techniques: Binding Assay, Selection