Cell culture and reagents

HeLa, HCT116, and NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM l‐glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37°C and 5% CO₂. RPE cells were maintained in DMEM:F12 supplemented with 10% fetal bovine serum (FBS), 2 mM L‐glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37°C and 5% CO₂. For HeLa‐H2B/tub cells 30, 1 mg/ml puromycin and 400 μg/ml G418 were added to the medium. For HeLa_dox‐YFP‐TIARr cells, 100 μg/ml of zeocin and 5 μg/ml of blasticidin were added to the medium. For synchronization, HeLa and HeLa‐H2B/tub cells were subjected to a double thymidine block following standard procedures (18 h 2 mM thymidine, 9 h release, and 18 h 2 mM thymidine). Nocodazole (Sigma) was used at 200 nM, APH (Sigma) at 0.4 μM, UCN‐01 (Sigma) at 300 nM, Gö6976 (Calbiochem) at 1 μg/ml, ATRi (ETP‐46464 9) at 4 μM, and ICRF‐193 (Sigma) at 1 μM.

The HeLa_dox‐YFP‐TIARr, HeLa_dox‐YFP‐TIARr‐RRM123m, and HeLa_dox‐YFP‐TIARr‐dQRD cell lines were generated by transfection of HeLa‐TREX cells 66 with pcDNA4/TO‐YFP‐TIARr (p3380), pcDNA4/TO‐YFP‐TIARr‐RRM123 m (p3414), and pcDNA4/TO‐YFP‐TIARr‐dQRD (p3321), respectively, using Lipofectamine 2000 (Invitrogen). Stably transfected cells were selected with 200 μg/ml of zeocin (Invitrogen) and 10 μg/ml of blasticidin (Invitrogen). After selection, cells were subcloned, and clones #5 (YFP‐TIARr), #11 (YFP‐TIARr‐RRM123 m), and #8 (YFP‐TIARr‐dQRD) were chosen. Clone #5 (YFP‐TIARr) was additionally sorted for YFP‐TIARr expressing cells by flow cytometry after adding 1 μg/ml of doxycycline for 18 h.