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. 2023 May 17;14(5):328. doi: 10.1038/s41419-023-05845-6

Fig. 3. DSBR pathway usage in cells expressing mutated versus wild-type ABRAXAS1.

Fig. 3

Repair events were analyzed by flow cytometry 48 h after transfection of LCLs with EGFP-based and pathway-specific DSBR reporter plasmids plus I-SceI meganuclease expression plasmid. Percentages of EGFP-positive cells in the live cell population were individually corrected for transfection efficiencies obtained in split culture samples transfected with the same plasmid mixture but replacing a filler plasmid by wild-type EGFP expression plasmid (triplicates for repair events and triplicates for transfection efficiencies each). Resulting DSBR frequencies were normalized to the mean frequencies for controls measured on the same day, namely for both wild-type ABRAXAS1 cell lines in (a) and for wild-type ABRAXAS1 cells transfected with empty vector in (b). These reference values were defined as 100% (corresponding to 3.7 × 10−4 for HR, 3.9 × 10−3 for NHEJ, 5.3 × 10−4 for MMEJ, 2.5 × 10−3 for SSA). Columns show DSBR frequencies; bars, SEM; GraphPad Prism software was used for graphic presentation and calculation of statistically significant differences via Kruskal–Wallis-test followed by two-tailed Mann–Whitney U test. *P < 0.05, **P < 0.01, **** P < 0.0001; a DSBR in heterozygously ABRAXAS1-mutated and wild-type individuals. LCLs from two wild-type ABRAXAS1 individuals (ABR-wt: white columns and external control BR-0968: hatched), one ABRAXAS1 c.1106dup (ABR-1106: light grey) and one ABRAXAS1 c.577C>T (ABR-577: dark grey) mutation carrier were transfected with a DNA mixture containing reporter constructs for analysis of HR, NHEJ, MMEJ or SSA (schematically drawn on the left side of each panel [80, 79]). Statistically significant differences were calculated between the mean values in ABR-wt, ABR-1106 and ABR-577. n = 15 from five independent experiments; b DSBR after ectopic expression of ABRAXAS1 variants. LCL ABR-wt cells were co-transfected with plasmid mixtures for HR, NHEJ or SSA measurements including I-SceI meganuclease expression plasmid (see a) plus expression plasmids for wild-type ABRAXAS1 (wt: white), the mutated variants (dup, ABRAXAS1 c.1106dup: light grey; c.577, ABRAXAS1 c.577C>T: dark grey) or empty vector (ctrl: hatched). Statistically significant differences were calculated between the mean values in cells expressing exogenous wild-type versus mutated ABRAXAS1. n = 6–9 from two to three independent experiments.