Fig. 6: O-GlcNAcylation of XRCC4 at Thr308 protects cancer cells against cell death in response to DNA double-strand break damage.

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).