Cell culture and reagents

HCT-116 (from ATCC), ZC3H4-mAID, ZC3H4-Turbo-ID, ZC3H4-mAID:SYMPK-dTAG, ZC3H4-mAID:PNUTS-dTAG, ZC3H4-Turbo-ID:SYMPK-dTAG, and ZC3H4-Turbo-ID:PNUTS-dTAG HCT-116 cells were cultured in McCoy's 5A medium modified with 10% South American serum, 1% penicillin/streptomycin (Sigma P4333), and 1% L-Glutamax (Gibco 35050061). HeLa TREx Flp-In cell lines were cultured in DMEM with 10% tetracycline-free serum (Euroclone ECS0182L), 1% penicillin/streptomycin, 250 µg/mL hygromycin B (Invitrogen 10687010), and 1 µg/mL blasticidin (Gibco R210-01). Cell lines were authenticated by the Tissue Culture Facility of the European Institute of Oncology using the GenePrint10 system (Promega) and routinely screened for mycoplasma contamination. For the streptavidin pull-down experiment, ZC3H4-Turbo-ID and wild-type HCT-116 cells were maintained in DMEM with 10% dialyzed fetal calf serum (Thermo Fisher 26400044) and 1% penicillin/streptomycin for 5 d. At day 5, 50 µM biotin (Sigma B4639) was added to the cells for 10 min. Auxin (Sigma I5148) was added at a final concentration of 100 µM for 1, 2, 4, or 24 h as indicated. dTAG-13 (Tocris Bioscience 6605) was added at a final concentration of 500 nM for 4 or 24 h. Tetracycline hydrochloride (Sigma T7660) was added for 48 h at a final concentration of 100 ng/mL. 4sU (Santa Cruz Biotechnologies sc-204628A) was added to the medium at a final concentration of 500 μM for 10 min for HCT-116 cells or 300 µM for 45 min for HeLa TREx Flp-In cells before harvesting.