Figure 4

ALDOA is required for DNA double-strand break repair. (A) Schematic of the DSB repair reporter assay used, repair by NHEJ or HR leads to GFP or mCherry expression, respectively²⁹ Repair of DSBs by NHEJ and HR were assayed using the U2OS DSB.3 U2OS cell line-based chromosomal reporter system, containing a single integrated GFP reporter with two I-SceI target sites in inverted orientation, flanking an out of frame ATG upstream of the GFP reporter. Cleavage by I-SceI removes the ATG, leaving non-compatible ends. Figure generated with Biorender.com. (B) U2OS cells containing the stably integrated HR/NHEJ reporter construct were depleted of ALDOA using siRNA and transfected with Flag or Flag-ALDOA, confirmed via immunoblot. (C,D) Depletion of ALDOA results in decreased NHEJ (C) and HR (D). Cells were transfected with control or ALDOA siRNA, then transfected with pcDNA3.1/I-SceI after 48 h. Cells transfected with empty Flag or siRNA resistant Flag-ALDOA showed the restoration of HR/NHEJ activity. Unless otherwise stated, data points represent the mean and the error bars represent the SD of three independent experiments and t-test was used for statistical analysis: *P < 0.05 **P < 0.01.