Research Paper Volume 13, Issue 17 pp 21066—21089

Sex-specific differences in DNA double-strand break repair of cycling human lymphocytes during aging

Analysis of BLM-dependent DSB repair and BLM foci formation in PBL from female donors. (A–C) DSB repair activity measurements. Cultivated PBL were nucleofected with a DNA mixture containing pCMV-I-SceI, repair substrate EJ5SceGFP (NHEJ) (A) or EJ-EGFP (MMEJ) (B), pBS or wild-type EGFP expression plasmid and knockdown (kd) plasmids silencing BLM or empty vector controls. Mean values for samples nucleofected with control plasmid were set to 100% for each donor. Columns, mean values; bars, SD; n=4-9 donors; *, pSupplementary Table 1) (C) quantitative PCR analysis of BLM expression to validate knockdown efficiency. Columns, mean relative expression; bars, SD; female: n=4 (young), n=2 (old). (D, E) BLM foci formation. BLM was immunocytochemically detected 24h post IR. Foci numbers of 50-200 nuclei per donor were scored. (D) Columns, mean values; bars, SEM; female: n=13 (young), n=11 (old) (Supplementary Table 1). (E) Representative immunofluorescence images of nuclei with IR-induced BLM foci.

Figure 5. Analysis of BLM-dependent DSB repair and BLM foci formation in PBL from female donors. (AC) DSB repair activity measurements. Cultivated PBL were nucleofected with a DNA mixture containing pCMV-I-SceI, repair substrate EJ5SceGFP (NHEJ) (A) or EJ-EGFP (MMEJ) (B), pBS or wild-type EGFP expression plasmid and knockdown (kd) plasmids silencing BLM or empty vector controls. Mean values for samples nucleofected with control plasmid were set to 100% for each donor. Columns, mean values; bars, SD; n=4-9 donors; *, p<0.05; Wilcoxon matched-pairs signed rank test (Supplementary Table 1) (C) quantitative PCR analysis of BLM expression to validate knockdown efficiency. Columns, mean relative expression; bars, SD; female: n=4 (young), n=2 (old). (D, E) BLM foci formation. BLM was immunocytochemically detected 24h post IR. Foci numbers of 50-200 nuclei per donor were scored. (D) Columns, mean values; bars, SEM; female: n=13 (young), n=11 (old) (Supplementary Table 1). (E) Representative immunofluorescence images of nuclei with IR-induced BLM foci.