Journal: Cell Death & Disease

Article Title: O -GlcNAcylation of XRCC4 controls its stability and confers resistance to DNA double-strand break damage in cancer cells

doi: 10.1038/s41419-025-08209-4

Figure Lengend Snippet: A Colon tumors were induced in mice using azoxymethane (AOM) and dextran sulfate sodium (DSS). The levels of O -GlcNAcylated proteins were assessed by RL2 blotting. The levels of cellular O -GlcNAcylation and XRCC4 protein were compared between normal colon tissue and colon tumor (red arrow), normalizing the expression to GAPDH levels ( n = 3). B Cellular O -GlcNAcylation and XRCC4 protein levels were compared between colon normal and cancer cell lines. XRCC4 expression was normalized to GAPDH levels ( n = 3). C – F Cellular O -GlcNAcylation levels were regulated by C OGT transfection ( n = 4), D 2 μM Thiamet-G (TMG), an OGA inhibitor, treatment ( n = 4), E OGA transfection ( n = 3), or F 30 μM OSMI-1, an OGT inhibitor, treatment ( n = 5). The cell lysates were subjected to immunoblotting with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels. G HCT116 cells were cultured in media containing 5 or 25 mM glucose for 24 h and transfected with control vector or OGT for 24 h. XRCC4 expression was normalized to GAPDH levels ( n = 4). A – G Data were represented as means ± SD. Statistical significance was determined by the two-tailed Student’s t -test or one-way ANOVA for multiple comparisons ( *P < 0.05, **P < 0.01, and ***P < 0.001).

Article Snippet: For immunoprecipitation of endogenous XRCC4, cell lysates were incubated with anti-XRCC4 antibody (Santa Cruz, #sc-271087, USA) and with agarose-conjugated protein A/G (Santa Cruz, #sc-2003, USA).

Techniques: Expressing, Transfection, Western Blot, Cell Culture, Control, Plasmid Preparation, Two Tailed Test

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation of XRCC4 controls its stability and confers resistance to DNA double-strand break damage in cancer cells

doi: 10.1038/s41419-025-08209-4

Figure Lengend Snippet: A Total RNA was extracted from HCT116 cells transfected with OGT or OGA for 24 h. XRCC4 mRNA levels were quantified by RT-qPCR and normalized to β-actin levels ( n = 5). B , C HCT116 cells were transfected with B OGT ( n = 5) or C OGA ( n = 5) for 24 h, followed by 200 μg/mL cycloheximide (CHX), a protein synthesis inhibitor, treatment. Cells were collected at the indicated times. XRCC4 degradation rates were assessed by immunoblotting. XRCC4 expression was normalized to GAPDH levels. D , E HCT116 cells were transfected with OGA for 24 h and treated with 20 μM MG132, a proteasome inhibitor, for 6 h. D The cell lysates were subjected to immunoblotting with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels ( n = 4). E For the ubiquitination assay, the cell lysates were immunoprecipitated with the XRCC4 antibody. The polyubiquitination levels of XRCC4 were analyzed by immunoblotting with K48-Ub antibody. A – D Data were represented as means ± SD. Statistical significance was determined by the two-tailed Student’s t -test or one-way ANOVA for multiple comparisons ( *P < 0.05, **P < 0.01, and ***P < 0.001).

Article Snippet: For immunoprecipitation of endogenous XRCC4, cell lysates were incubated with anti-XRCC4 antibody (Santa Cruz, #sc-271087, USA) and with agarose-conjugated protein A/G (Santa Cruz, #sc-2003, USA).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Expressing, Ubiquitin Proteomics, Immunoprecipitation, Two Tailed Test

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation of XRCC4 controls its stability and confers resistance to DNA double-strand break damage in cancer cells

doi: 10.1038/s41419-025-08209-4

Figure Lengend Snippet: A Table showing the candidates of E3 ubiquitin ligases for XRCC4 as predicted by analyzing the interactome of XRCC4. B HCT116 cells were transfected with TRIM28 or TRIM21 siRNAs for 48 h. The cell lysates were immunoblotted with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels ( n = 3). C HCT116 cells were transfected with FLAG-TRIM28 ( n = 4) or Myc-TRIM21 ( n = 6) for 24 h. The cell lysates were immunoblotted with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels. D HCT116 cells were transfected with TRIM21 siRNA for 48 h and treated with 20 μM MG132 for 6 h. For the ubiquitination assay, the cell lysates were immunoprecipitated with the indicated antibodies. The polyubiquitination levels of XRCC4 were analyzed by immunoblotting with K48-Ub antibody. E HCT116 cells were transfected with TRIM21 siRNA for 48 h and with OGA for 24 h. The cell lysates were subjected to immunoblotting with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels ( n = 3). F HCT116 cells were transfected with OGA for 24 h. For co-immunoprecipitation, the cell lysates were immunoprecipitated with the XRCC4 antibody. The affinity levels between XRCC4 and TRIM21 were analyzed by immunoblotting with the TRIM21 antibody. Co-immunoprecipitated TRIM21 levels were normalized to immunoprecipitated XRCC4 levels ( n = 3). B , C , E , F Data were represented as means ± SD. Statistical significance was determined by the two-tailed Student’s t -test or one-way ANOVA for multiple comparisons ( **P < 0.01, ***P < 0.001, ns not significant).

Article Snippet: For immunoprecipitation of endogenous XRCC4, cell lysates were incubated with anti-XRCC4 antibody (Santa Cruz, #sc-271087, USA) and with agarose-conjugated protein A/G (Santa Cruz, #sc-2003, USA).

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Immunoprecipitation, Western Blot, Two Tailed Test

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation of XRCC4 controls its stability and confers resistance to DNA double-strand break damage in cancer cells

doi: 10.1038/s41419-025-08209-4

Figure Lengend Snippet: A HCT116 cells were transfected with FLAG-XRCC4 or Myc-OGT for 24 h. The cell lysates were immunoprecipitated using FLAG beads. Exogenous O -GlcNAcylation of XRCC4 was detected by immunoblotting with RL2 antibody. For co-immunoprecipitation, the immunoprecipitated lysates were immunoblotted with Myc antibody. B , C HCT116 cells were transfected with B Myc-OGT or C V5-OGA for 24 h. The cell lysates were immunoprecipitated using the XRCC4 antibody. Endogenous O -GlcNAcylation of XRCC4 was detected by immunoblotting with RL2 antibody. B For co-immunoprecipitation, the immunoprecipitated lysates were immunoblotted with Myc antibody. D Putative O -GlcNAc sites of XRCC4 were identified by EThcD-MS/MS. O -GlcNAcylated XRCC4 peptides, 297-ENSRPDSSLPEtSK-310, is shown. E A schematic drawing of the putative XRCC4 O -GlcNAcylation sites, including Ser303, Ser304, Thr308, and Ser309, based on mass spectrometry data. F HCT116 cells were transiently transfected with each indicated FLAG-XRCC4 point mutant. The cell lysates were immunoprecipitated using FLAG beads. O -GlcNAcylation of each FLAG-XRCC4 point mutant was detected by immunoblotting with RL2 antibody. O -GlcNAcylated FLAG-XRCC4 was normalized to immunoprecipitated FLAG-XRCC4 ( n = 4). Data were represented as means ± SD. Statistical significance was determined by the two-tailed Student’s t -test ( ***P < 0.001). G Cross-species sequence alignment of XRCC4.

Article Snippet: For immunoprecipitation of endogenous XRCC4, cell lysates were incubated with anti-XRCC4 antibody (Santa Cruz, #sc-271087, USA) and with agarose-conjugated protein A/G (Santa Cruz, #sc-2003, USA).

Techniques: Transfection, Immunoprecipitation, Western Blot, Tandem Mass Spectroscopy, Mass Spectrometry, Mutagenesis, Two Tailed Test, Sequencing

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation of XRCC4 controls its stability and confers resistance to DNA double-strand break damage in cancer cells

doi: 10.1038/s41419-025-08209-4

Figure Lengend Snippet: A FLAG-XRCC4 WT or T308A were stably expressed in HCT116 XRCC4 KO cells. The cell lysates were immunoblotted with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels ( n = 3). B Total RNA was extracted from HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4. XRCC4 mRNA levels were quantified by RT-qPCR and normalized to β-actin levels ( n = 4). C HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were treated with 200 μg/mL cycloheximide (CHX). Cells were collected every 24 h and the cell lysates were immunoblotted with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels ( n = 4). D HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were treated with 20 μM MG132 for 6 h. For the ubiquitination assay, the cell lysates were immunoprecipitated using the XRCC4 antibody. The polyubiquitination levels of XRCC4 were analyzed by immunoblotting with K48-Ub antibody. E For co-immunoprecipitation, the cell lysates from HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were subjected to immunoprecipitation with XRCC4 antibody. The affinity levels between XRCC4 and TRIM21 were analyzed by immunoblotting with the TRIM21 antibody. TRIM21 expression was normalized to GAPDH levels ( n = 3). Co-immunoprecipitated TRIM21 levels were normalized to immunoprecipitated XRCC4 levels ( n = 3). A – C , E Data were represented as means ± SD. Statistical significance was determined by the two-tailed Student’s t -test ( **P < 0.01).

Article Snippet: For immunoprecipitation of endogenous XRCC4, cell lysates were incubated with anti-XRCC4 antibody (Santa Cruz, #sc-271087, USA) and with agarose-conjugated protein A/G (Santa Cruz, #sc-2003, USA).

Techniques: Stable Transfection, Expressing, Quantitative RT-PCR, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Two Tailed Test

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation of XRCC4 controls its stability and confers resistance to DNA double-strand break damage in cancer cells

doi: 10.1038/s41419-025-08209-4

Figure Lengend Snippet: A , B HCT116 cells were treated with 10 μg/mL bleomycin. Cells were collected with the indicated time. A The cell lysates were immunoblotted with the indicated antibodies. XRCC4 and O -GlcNAcylation levels were normalized to GAPDH levels ( n = 4). B Endogenous O -GlcNAcylation of XRCC4 at the indicated time was detected by immunoblotting with RL2 antibody. O -GlcNAcylated XRCC4 was normalized to immunoprecipitated XRCC4 ( n = 3). C Total RNA was extracted from HCT116 cells treated with or without 10 μg/mL bleomycin for 24 h. XRCC4 mRNA levels were quantified by RT-qPCR and normalized to β-actin levels ( n = 5). D HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were treated with or without 10 μg/mL bleomycin for 24 h. The cell lysates were immunoblotted with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels ( n = 5). E HCT116 XRCC4 knockout (KO) cells stably expressing either wild-type (WT) or T308A mutant XRCC4 were treated with 10 μg/mL bleomycin. Cells were harvested at the indicated time points, and whole-cell lysates were subjected to immunoblotting with the specified antibodies. XRCC4 and γH2AX expression levels were normalized to GAPDH. The bar graph depicts relative γH2AX levels at 24 and 48 h in HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 ( n = 3). F XRCC4 was knocked down using siRNA and treated with or without 10 μg/mL bleomycin. WST-8 assay was performed after treatment with bleomycin for the indicated time. Quantification of relative growth rates was performed at 96 h ( n = 4). G HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were all treated with 10 μg/mL bleomycin and were transfected with either control vector or OGA. WST-8 assay was performed after treatment with bleomycin for the indicated time ( n = 5). H HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were treated with or without 10 μg/mL bleomycin. WST-8 assay was performed after treatment with bleomycin for the indicated time ( n = 4). A – H Data were represented as means ± SD. Statistical significance was determined by the two-tailed Student’s t -test ( *P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: For immunoprecipitation of endogenous XRCC4, cell lysates were incubated with anti-XRCC4 antibody (Santa Cruz, #sc-271087, USA) and with agarose-conjugated protein A/G (Santa Cruz, #sc-2003, USA).

Techniques: Western Blot, Immunoprecipitation, Quantitative RT-PCR, Stable Transfection, Expressing, Knock-Out, Mutagenesis, Transfection, Control, Plasmid Preparation, Two Tailed Test

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation of XRCC4 controls its stability and confers resistance to DNA double-strand break damage in cancer cells

doi: 10.1038/s41419-025-08209-4

Figure Lengend Snippet: A HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were transfected with OGA. WST-8 assay was performed after growth for the indicated time ( n = 5). B HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were subject to a Transwell invasion assay. Invaded cells were measured by WST-8 assay ( n = 7). C HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were subject to colony formation assays. Colony formation was quantified by using a 485/520 nm filter set ( n = 8). D Each group of BALB/c nude mice ( n = 6 per group) were injected with 1 × 10 7 of the mentioned cells into the hypodermis. Tumor size and weight were quantified 60 days after injection. A – D Data were represented as means ± SD. Statistical significance was determined by the two-tailed Student’s t -test or one-way ANOVA for multiple comparisons ( **P < 0.01, ***P < 0.001).

Article Snippet: For immunoprecipitation of endogenous XRCC4, cell lysates were incubated with anti-XRCC4 antibody (Santa Cruz, #sc-271087, USA) and with agarose-conjugated protein A/G (Santa Cruz, #sc-2003, USA).

Techniques: Stable Transfection, Expressing, Transfection, Transwell Invasion Assay, Injection, Two Tailed Test

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation of XRCC4 controls its stability and confers resistance to DNA double-strand break damage in cancer cells

doi: 10.1038/s41419-025-08209-4

Figure Lengend Snippet: O -GlcNAcylation of XRCC4 at Thr308 increases the stability of the protein by inhibiting the interaction between XRCC4 and the E3 ligase TRIM21. Upregulated XRCC4 promotes resistance to DNA double-strand breaks and cancer cell proliferation.

Article Snippet: For immunoprecipitation of endogenous XRCC4, cell lysates were incubated with anti-XRCC4 antibody (Santa Cruz, #sc-271087, USA) and with agarose-conjugated protein A/G (Santa Cruz, #sc-2003, USA).

Techniques:

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation of XRCC4 controls its stability and confers resistance to DNA double-strand break damage in cancer cells

doi: 10.1038/s41419-025-08209-4

Figure Lengend Snippet: A Colon tumors were induced in mice using azoxymethane (AOM) and dextran sulfate sodium (DSS). The levels of O -GlcNAcylated proteins were assessed by RL2 blotting. The levels of cellular O -GlcNAcylation and XRCC4 protein were compared between normal colon tissue and colon tumor (red arrow), normalizing the expression to GAPDH levels ( n = 3). B Cellular O -GlcNAcylation and XRCC4 protein levels were compared between colon normal and cancer cell lines. XRCC4 expression was normalized to GAPDH levels ( n = 3). C – F Cellular O -GlcNAcylation levels were regulated by C OGT transfection ( n = 4), D 2 μM Thiamet-G (TMG), an OGA inhibitor, treatment ( n = 4), E OGA transfection ( n = 3), or F 30 μM OSMI-1, an OGT inhibitor, treatment ( n = 5). The cell lysates were subjected to immunoblotting with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels. G HCT116 cells were cultured in media containing 5 or 25 mM glucose for 24 h and transfected with control vector or OGT for 24 h. XRCC4 expression was normalized to GAPDH levels ( n = 4). A – G Data were represented as means ± SD. Statistical significance was determined by the two-tailed Student’s t -test or one-way ANOVA for multiple comparisons ( *P < 0.05, **P < 0.01, and ***P < 0.001).

Article Snippet: The samples were then diluted and rotated at 4 °C for 30 min. After centrifugation at 13,000 rpm for 20 min, the supernatants were subjected to immunoprecipitation with an XRCC4 antibody (Santa Cruz Biotechnology) overnight at 4 °C.

Techniques: Expressing, Transfection, Western Blot, Cell Culture, Control, Plasmid Preparation, Two Tailed Test

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation of XRCC4 controls its stability and confers resistance to DNA double-strand break damage in cancer cells

doi: 10.1038/s41419-025-08209-4

Figure Lengend Snippet: A Total RNA was extracted from HCT116 cells transfected with OGT or OGA for 24 h. XRCC4 mRNA levels were quantified by RT-qPCR and normalized to β-actin levels ( n = 5). B , C HCT116 cells were transfected with B OGT ( n = 5) or C OGA ( n = 5) for 24 h, followed by 200 μg/mL cycloheximide (CHX), a protein synthesis inhibitor, treatment. Cells were collected at the indicated times. XRCC4 degradation rates were assessed by immunoblotting. XRCC4 expression was normalized to GAPDH levels. D , E HCT116 cells were transfected with OGA for 24 h and treated with 20 μM MG132, a proteasome inhibitor, for 6 h. D The cell lysates were subjected to immunoblotting with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels ( n = 4). E For the ubiquitination assay, the cell lysates were immunoprecipitated with the XRCC4 antibody. The polyubiquitination levels of XRCC4 were analyzed by immunoblotting with K48-Ub antibody. A – D Data were represented as means ± SD. Statistical significance was determined by the two-tailed Student’s t -test or one-way ANOVA for multiple comparisons ( *P < 0.05, **P < 0.01, and ***P < 0.001).

Article Snippet: The samples were then diluted and rotated at 4 °C for 30 min. After centrifugation at 13,000 rpm for 20 min, the supernatants were subjected to immunoprecipitation with an XRCC4 antibody (Santa Cruz Biotechnology) overnight at 4 °C.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Expressing, Ubiquitin Proteomics, Immunoprecipitation, Two Tailed Test

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation of XRCC4 controls its stability and confers resistance to DNA double-strand break damage in cancer cells

doi: 10.1038/s41419-025-08209-4

Figure Lengend Snippet: A Table showing the candidates of E3 ubiquitin ligases for XRCC4 as predicted by analyzing the interactome of XRCC4. B HCT116 cells were transfected with TRIM28 or TRIM21 siRNAs for 48 h. The cell lysates were immunoblotted with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels ( n = 3). C HCT116 cells were transfected with FLAG-TRIM28 ( n = 4) or Myc-TRIM21 ( n = 6) for 24 h. The cell lysates were immunoblotted with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels. D HCT116 cells were transfected with TRIM21 siRNA for 48 h and treated with 20 μM MG132 for 6 h. For the ubiquitination assay, the cell lysates were immunoprecipitated with the indicated antibodies. The polyubiquitination levels of XRCC4 were analyzed by immunoblotting with K48-Ub antibody. E HCT116 cells were transfected with TRIM21 siRNA for 48 h and with OGA for 24 h. The cell lysates were subjected to immunoblotting with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels ( n = 3). F HCT116 cells were transfected with OGA for 24 h. For co-immunoprecipitation, the cell lysates were immunoprecipitated with the XRCC4 antibody. The affinity levels between XRCC4 and TRIM21 were analyzed by immunoblotting with the TRIM21 antibody. Co-immunoprecipitated TRIM21 levels were normalized to immunoprecipitated XRCC4 levels ( n = 3). B , C , E , F Data were represented as means ± SD. Statistical significance was determined by the two-tailed Student’s t -test or one-way ANOVA for multiple comparisons ( **P < 0.01, ***P < 0.001, ns not significant).

Article Snippet: The samples were then diluted and rotated at 4 °C for 30 min. After centrifugation at 13,000 rpm for 20 min, the supernatants were subjected to immunoprecipitation with an XRCC4 antibody (Santa Cruz Biotechnology) overnight at 4 °C.

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Immunoprecipitation, Western Blot, Two Tailed Test

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation of XRCC4 controls its stability and confers resistance to DNA double-strand break damage in cancer cells

doi: 10.1038/s41419-025-08209-4

Figure Lengend Snippet: A HCT116 cells were transfected with FLAG-XRCC4 or Myc-OGT for 24 h. The cell lysates were immunoprecipitated using FLAG beads. Exogenous O -GlcNAcylation of XRCC4 was detected by immunoblotting with RL2 antibody. For co-immunoprecipitation, the immunoprecipitated lysates were immunoblotted with Myc antibody. B , C HCT116 cells were transfected with B Myc-OGT or C V5-OGA for 24 h. The cell lysates were immunoprecipitated using the XRCC4 antibody. Endogenous O -GlcNAcylation of XRCC4 was detected by immunoblotting with RL2 antibody. B For co-immunoprecipitation, the immunoprecipitated lysates were immunoblotted with Myc antibody. D Putative O -GlcNAc sites of XRCC4 were identified by EThcD-MS/MS. O -GlcNAcylated XRCC4 peptides, 297-ENSRPDSSLPEtSK-310, is shown. E A schematic drawing of the putative XRCC4 O -GlcNAcylation sites, including Ser303, Ser304, Thr308, and Ser309, based on mass spectrometry data. F HCT116 cells were transiently transfected with each indicated FLAG-XRCC4 point mutant. The cell lysates were immunoprecipitated using FLAG beads. O -GlcNAcylation of each FLAG-XRCC4 point mutant was detected by immunoblotting with RL2 antibody. O -GlcNAcylated FLAG-XRCC4 was normalized to immunoprecipitated FLAG-XRCC4 ( n = 4). Data were represented as means ± SD. Statistical significance was determined by the two-tailed Student’s t -test ( ***P < 0.001). G Cross-species sequence alignment of XRCC4.

Article Snippet: The samples were then diluted and rotated at 4 °C for 30 min. After centrifugation at 13,000 rpm for 20 min, the supernatants were subjected to immunoprecipitation with an XRCC4 antibody (Santa Cruz Biotechnology) overnight at 4 °C.

Techniques: Transfection, Immunoprecipitation, Western Blot, Tandem Mass Spectroscopy, Mass Spectrometry, Mutagenesis, Two Tailed Test, Sequencing

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation of XRCC4 controls its stability and confers resistance to DNA double-strand break damage in cancer cells

doi: 10.1038/s41419-025-08209-4

Figure Lengend Snippet: A FLAG-XRCC4 WT or T308A were stably expressed in HCT116 XRCC4 KO cells. The cell lysates were immunoblotted with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels ( n = 3). B Total RNA was extracted from HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4. XRCC4 mRNA levels were quantified by RT-qPCR and normalized to β-actin levels ( n = 4). C HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were treated with 200 μg/mL cycloheximide (CHX). Cells were collected every 24 h and the cell lysates were immunoblotted with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels ( n = 4). D HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were treated with 20 μM MG132 for 6 h. For the ubiquitination assay, the cell lysates were immunoprecipitated using the XRCC4 antibody. The polyubiquitination levels of XRCC4 were analyzed by immunoblotting with K48-Ub antibody. E For co-immunoprecipitation, the cell lysates from HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were subjected to immunoprecipitation with XRCC4 antibody. The affinity levels between XRCC4 and TRIM21 were analyzed by immunoblotting with the TRIM21 antibody. TRIM21 expression was normalized to GAPDH levels ( n = 3). Co-immunoprecipitated TRIM21 levels were normalized to immunoprecipitated XRCC4 levels ( n = 3). A – C , E Data were represented as means ± SD. Statistical significance was determined by the two-tailed Student’s t -test ( **P < 0.01).

Article Snippet: The samples were then diluted and rotated at 4 °C for 30 min. After centrifugation at 13,000 rpm for 20 min, the supernatants were subjected to immunoprecipitation with an XRCC4 antibody (Santa Cruz Biotechnology) overnight at 4 °C.

Techniques: Stable Transfection, Expressing, Quantitative RT-PCR, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Two Tailed Test

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation of XRCC4 controls its stability and confers resistance to DNA double-strand break damage in cancer cells

doi: 10.1038/s41419-025-08209-4

Figure Lengend Snippet: A , B HCT116 cells were treated with 10 μg/mL bleomycin. Cells were collected with the indicated time. A The cell lysates were immunoblotted with the indicated antibodies. XRCC4 and O -GlcNAcylation levels were normalized to GAPDH levels ( n = 4). B Endogenous O -GlcNAcylation of XRCC4 at the indicated time was detected by immunoblotting with RL2 antibody. O -GlcNAcylated XRCC4 was normalized to immunoprecipitated XRCC4 ( n = 3). C Total RNA was extracted from HCT116 cells treated with or without 10 μg/mL bleomycin for 24 h. XRCC4 mRNA levels were quantified by RT-qPCR and normalized to β-actin levels ( n = 5). D HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were treated with or without 10 μg/mL bleomycin for 24 h. The cell lysates were immunoblotted with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels ( n = 5). E HCT116 XRCC4 knockout (KO) cells stably expressing either wild-type (WT) or T308A mutant XRCC4 were treated with 10 μg/mL bleomycin. Cells were harvested at the indicated time points, and whole-cell lysates were subjected to immunoblotting with the specified antibodies. XRCC4 and γH2AX expression levels were normalized to GAPDH. The bar graph depicts relative γH2AX levels at 24 and 48 h in HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 ( n = 3). F XRCC4 was knocked down using siRNA and treated with or without 10 μg/mL bleomycin. WST-8 assay was performed after treatment with bleomycin for the indicated time. Quantification of relative growth rates was performed at 96 h ( n = 4). G HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were all treated with 10 μg/mL bleomycin and were transfected with either control vector or OGA. WST-8 assay was performed after treatment with bleomycin for the indicated time ( n = 5). H HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were treated with or without 10 μg/mL bleomycin. WST-8 assay was performed after treatment with bleomycin for the indicated time ( n = 4). A – H Data were represented as means ± SD. Statistical significance was determined by the two-tailed Student’s t -test ( *P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The samples were then diluted and rotated at 4 °C for 30 min. After centrifugation at 13,000 rpm for 20 min, the supernatants were subjected to immunoprecipitation with an XRCC4 antibody (Santa Cruz Biotechnology) overnight at 4 °C.

Techniques: Western Blot, Immunoprecipitation, Quantitative RT-PCR, Stable Transfection, Expressing, Knock-Out, Mutagenesis, Transfection, Control, Plasmid Preparation, Two Tailed Test

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation of XRCC4 controls its stability and confers resistance to DNA double-strand break damage in cancer cells

doi: 10.1038/s41419-025-08209-4

Figure Lengend Snippet: A HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were transfected with OGA. WST-8 assay was performed after growth for the indicated time ( n = 5). B HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were subject to a Transwell invasion assay. Invaded cells were measured by WST-8 assay ( n = 7). C HCT116 XRCC4 KO cells stably expressing WT or T308A XRCC4 were subject to colony formation assays. Colony formation was quantified by using a 485/520 nm filter set ( n = 8). D Each group of BALB/c nude mice ( n = 6 per group) were injected with 1 × 10 7 of the mentioned cells into the hypodermis. Tumor size and weight were quantified 60 days after injection. A – D Data were represented as means ± SD. Statistical significance was determined by the two-tailed Student’s t -test or one-way ANOVA for multiple comparisons ( **P < 0.01, ***P < 0.001).

Article Snippet: The samples were then diluted and rotated at 4 °C for 30 min. After centrifugation at 13,000 rpm for 20 min, the supernatants were subjected to immunoprecipitation with an XRCC4 antibody (Santa Cruz Biotechnology) overnight at 4 °C.

Techniques: Stable Transfection, Expressing, Transfection, Transwell Invasion Assay, Injection, Two Tailed Test

Journal: Cell Death & Disease

Article Title: O -GlcNAcylation of XRCC4 controls its stability and confers resistance to DNA double-strand break damage in cancer cells

doi: 10.1038/s41419-025-08209-4

Figure Lengend Snippet: O -GlcNAcylation of XRCC4 at Thr308 increases the stability of the protein by inhibiting the interaction between XRCC4 and the E3 ligase TRIM21. Upregulated XRCC4 promotes resistance to DNA double-strand breaks and cancer cell proliferation.

Article Snippet: The samples were then diluted and rotated at 4 °C for 30 min. After centrifugation at 13,000 rpm for 20 min, the supernatants were subjected to immunoprecipitation with an XRCC4 antibody (Santa Cruz Biotechnology) overnight at 4 °C.

Techniques: