Ucn 01

All cell lines were kept at 37° C and 5% CO₂. 293T cells and JIMT-1 cells were grown in DMEM (Corning) supplemented with 10% fetal bovine serum (FBS, Gibco). SMF cells were grown in DMEM (Corning) supplemented with 10% FBS and 5 mg/ml insulin (Gemini Bio-Products). All media contained 2 mM glutamine (Corning) and penicillin/streptomycin (Corning), unless stated otherwise. Lapatinib was purchased from Santa Cruz Biotech. Axitinib, Dovitinib, KW-2449, staurosporine, UCN-01, midostaurin, lestaurtinib, PHA-665752, SU-14913, and sunitinib were purchased from Selleck Chemicals.

DNA combing data using the Xenopus in vitro system from Platel et al. 2015 [35 (link)] were analysed. Briefly, sperm nuclei (2000 nuclei/ $\mathsf{\mu }$ L) were incubated in Xenopus egg extracts in the presence of cycloheximide, energy mix and 20 $\mathsf{\mu }$ M biotin-dUTP (Roche Applied Science). UCN-01 (Selleck Chemicals) (or solvent (DMSO) alone as control) was added at 1 $\mathsf{\mu }$ M. Replication was allowed to continue for 40, 60 or 75 min. In order to increase the number of eye-to-eye distances in control samples at 40 min of the first experiment, data from two additional independent experiments with nearly identical replication content were combined for early S phase (45 and 50 min, respectively) (control 8%, UCN-01 22% replication.) For Chk1 overexpression experiments recombinant and active XChk1 with a N-terminal His-tag was expressed in the baculovirus expression system as described in Platel et al. 2015 [35 (link)]. We chose to moderately overexpress XChk1 threefold by adding 120 nM purified XChk1 (or dialysis buffer as control) in two independent replication reactions, which were stopped at 45 or 55 min, respectively. Labelled DNA was purified and DNA combing was performed as described in Platel et al., 2015 [35 (link)].