Fig. 1

Fig. 1

SPRTN depleted cells endure severe DNA replication stress but fail to activate a CHK1 response. a Schematic representation of DNA fiber assay. See also Methods. b DNA fibers obtained from HEK293 cells that have been treated with the indicated siRNAs against SPRTN (siSPRTN) or with hydroxyurea (HU). Scale bar: 10 µm. Data shown are representative images of three independent experiments. c–e DNA fiber assay analysis showing replication fork length (c), stalled replication forks (d) and newly fired origins (e). c siSPRTN cells exhibit decreased fork velocity. >100 individual IdU tracts were measured per experiment per condition. Data are shown as mean with 25–75% percentile range (box) and 10–90% percentile (whiskers); n = 3 independent experiments, two-tailed Student's t-test. d siSPRTN cells exhibit an increased frequency of stalled replication forks. >400 forks were scored per condition per experiment. Mean ± SEM; n = 3 independent experiments, two-tailed Student's t-test. e siSPRTN cells exhibit increased newly firing of dormant origin, contrary to cells treated with HU. >400 forks were scored per condition per experiment. Mean ± SEM; n = 3 independent experiments, two-tailed Student's t-test. f Knock-down of SPRTN in HEK293 cells diminishes the phosphorylation status of CHK1 residues S296 and S345, and enhances Cdc25A stability, contrary to treatment with HU. Immunoblots were run with whole cell lysates and represent three independent experiments. g Quantifications for (f) of CHK1 phosphorylation signal at residues S345 and S296, normalised to vinculin. Mean ± SEM; n = 3 independent experiments, two-tailed Student's t-test. h–j Treatment of cells with the CHK1 inhibitor UCN-01 induces severe replication stress to a similar extent as SPRTN-inactivation, detected by DNA fiber assay analysis. Schematic representation of experimental layout showing the addition of UCN-01 with IdU is also shown in (h, top); d: day. k Cell growth of wt and SPRTN-deficient (ΔSPRTN) HeLa cells in response to the CHK1 inhibitor UCN-01 (300 nM) on day 4 after seeding cells at same density (day 0). Mean ± SEM; n = 3 independent experiments, two-tailed Student's t-test. Source data for (c–k) are provided as a Source Data file