3-aminopropanal treatment accelerates dropout and increases DNA damage in FANCD2 ^−/− ALDH9A1 ^−/− cells. (A) A flow-based competition assay using WT and two independent FANCD2^−/− clones. As shown in Fig. 1 G, targeting by sgALDH9A1 results in a preferential dropout of two independent FANCD2^−/− clones. The rate of dropout was accelerated by the repeated treatment of 3-aminopropanal 12.5 µM (every 4 days). There was no apparent acceleration of dropout in the spermine-treated condition, in which cells were treated by spermine 250 µM and every 4 days from the day of mixing until day 15, after which the dose of spermine was increased to 500 µM until the end of the experiment. For the 3-aminopropanal treatment, two independent experiments were performed, and a representative figure is shown. For the spermine treatment, one experiment was performed in biological triplicate. Error bars, SD. N = 3. (B) γ-H2AX, H2AX, and vinculin western blot of Jurkat clones treated with or without 25 µM 3-aminopropanal for 24 h. (C) γ-H2AX, H2AX, and α-tubulin western blot of Jurkat clones treated with or without 250 µM spermine in 10% human serum-containing media for 72 h 3-APAL, 3-aminopropanal. Source data are available for this figure: SourceData F6.