Efficient TOE1 Depletion in HEK 293 Cells

All cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% Fetal Bovine Serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco) at 37 °C, 5% CO₂. Mycoplasma testing was routinely performed. TOE1 was depleted in HEK 293 T-REx-derived TOE1-degron cells (5 (link)) by incubation with 600 μM auxin hormone Indole-3-Acetic Acid (IAA, Sigma) for 8 h. RNA interference was performed in HEK 293 T Flp-In cells (FIRT, Thermo Fisher) with 20 nM small interfering (si)RNA targeting genes of interest, or luciferase as a control (SI Appendix, Table S1), using siLentFect (Bio-Rad) transfection reagent according to the manufacturer’s recommendations at 72 and 24 h before cell harvest. Plasmid transfections were performed using 2 μg plasmid per 3.5-cm well plates using Lipofectamine 2000 (Life Technologies) transfection reagent according to the manufacturer’s recommendations at 48 h before harvest, unless specified otherwise.