Figure 3
Figure 3. BRCA2Δexons 4-7 variant is efficient in DNA-repair function. (A) Methylene blue staining of HATr ES cell colonies expressing WT BRCA2, IVS7 + 2T > G BRCA2, and BRCA2Δexons 4-7. (B) Expression of BRCA2Δexons 4-7 variant analyzed by WB. Two independent clones were used for the mutant cell line. Actin was used as a control. (C) Sensitivity of 2 independent ES cell clones expressing BRCA2Δexons 4-7 to different DNA-damaging agents: mitomycin C (MMC); methyl-methanesulfonate (MMS); methyl-N′-nitro-N-nitrosoguanidine (MNNG); cisplatin; and ionizing radiation (IR). No, same as control (ES cells expressing WT BRCA2). (D) RAD51 foci formation 6 hours after ionizing radiation. RAD51 foci are shown in yellow, γ-H2AX foci marking DNA damage are shown in red, nuclei are stained with 4,6-diamidino-2-phenylindole (blue). Arrows point to RAD51 foci. Graphs on the right represent the quantification of RAD51 and γ-H2AX foci after ionizing radiation. Thirty nuclei were counted in each case, and their mean values are shown. Error bars represent the mean ± SD. (E) HR efficiency test. A scheme for targeting intron 1 of the Rosa26 locus (top panel). Number 1 indicates the first exon of Rosa26. WT indicates wild-type. Southern blot shows the targeted allele and WT allele. The bar graph on the right shows the quantification of the homologous recombination efficiency. Two independent clones were tested for BRCA2Δexons 4-7, and they are numbered as 1 and 2.

BRCA2Δexons 4-7 variant is efficient in DNA-repair function. (A) Methylene blue staining of HATr ES cell colonies expressing WT BRCA2, IVS7 + 2T > G BRCA2, and BRCA2Δexons 4-7. (B) Expression of BRCA2Δexons 4-7 variant analyzed by WB. Two independent clones were used for the mutant cell line. Actin was used as a control. (C) Sensitivity of 2 independent ES cell clones expressing BRCA2Δexons 4-7 to different DNA-damaging agents: mitomycin C (MMC); methyl-methanesulfonate (MMS); methyl-N′-nitro-N-nitrosoguanidine (MNNG); cisplatin; and ionizing radiation (IR). No, same as control (ES cells expressing WT BRCA2). (D) RAD51 foci formation 6 hours after ionizing radiation. RAD51 foci are shown in yellow, γ-H2AX foci marking DNA damage are shown in red, nuclei are stained with 4,6-diamidino-2-phenylindole (blue). Arrows point to RAD51 foci. Graphs on the right represent the quantification of RAD51 and γ-H2AX foci after ionizing radiation. Thirty nuclei were counted in each case, and their mean values are shown. Error bars represent the mean ± SD. (E) HR efficiency test. A scheme for targeting intron 1 of the Rosa26 locus (top panel). Number 1 indicates the first exon of Rosa26. WT indicates wild-type. Southern blot shows the targeted allele and WT allele. The bar graph on the right shows the quantification of the homologous recombination efficiency. Two independent clones were tested for BRCA2Δexons 4-7, and they are numbered as 1 and 2.

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