HR reporter assay.

Cells were transfected with the HR reporter DR-GFP plasmid using Lipofectamine 2000 (Invitrogen) following the manufacturer’s recommendations, with Geneticin (Thermo Fisher Scientific) selection and validation by flow cytometry for GFP⁺ cells after I-SceI treatment (58). Validated clones were plated in a 6-well plate, pretreated with the indicated siRNA for 24 hours, drug for 1 hour, or TTK plasmids for 48 hours. The SceI adenovirus was added to the cells for 48 hours, and cells were harvested and sorted by flow cytometry for GFP⁺ cells, which indicated successful HR.