Hemagglutinin (HA)–tagged WT, the arginine to lysine 111/113 mutant E2F1 (KK), and the arginine to lysine 109 (R109K) constructs have been described previously (11). These were subcloned into a pTRE2-hyg expression vector (Clontech) and transfected into parental Tet-On U2OS cells (Clontech; RRID: CVCL_V335) to generate inducible, stable cell lines. These cells were selected in Dulbecco’s modified Eagle medium (DMEM) (Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin/streptomycin, G418 (100 μg/ml; Santa Cruz Biotechnology), and hygromycin B (150 μg/ml; TOKU-E). For all experiments, doxycycline (1 μg/ml) was used to induce protein expression for 24 hours before harvest. E2F1 and TSN CRISPR cells were generated as per the protocol described (28) and cultured in DMEM containing 10% (v/v) FBS and penicillin/streptomycin. All cell lines were tested for mycoplasma contamination before use.

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