Fig. 3

Fig. 3

SPRTN does not contribute to the activation of CHK1 after DSB formation. a Phosphorylation status of the ATR-CHK1 and ATM-CHK2 pathways in control (siNS) and siRNA SPRTN-depleted HEK293 cells under unchallenged condition or when cells were treated with hydroxyurea (HU). Whole cell extracts were used for the immunoblots. b DNA fiber assay analysis comparing HeLa-wt (wild type, parental) and HeLa-ΔSPRTN cells: quantification of replication fork velocity (top; mean ± 25–75 percentile range (box) ± 10–90 percentile range (whiskers)), newly fired origins (bottom left; mean ± SEM) and stalled replication forks (bottom right; mean ± SEM). >100 DNA fibers were analysed per condition and experiment; n = 3 experimental replicates, two-tailed Student's t-test. c Analysis of the phosphorylation status of the ATR-CHK1 and ATM-CHK2 pathways comparing HeLa -wt and -ΔSPRTN under unchallenged or HU-treated conditions. Whole cell extracts were used for the immunoblots. SE: short exposure; LE: long exposure. d, e SPRTN-deficient (siSPRTN) or wt cells (siNS) treated with formaldehyde (FA; 50 μM, 1 h) do not exhibit robust single-stranded DNA foci in S-phase cells (CldU positive). HU (500 μM, 1 h) was used as a positive control to generate replication stress-induced ssDNA formation. Camptothecin (CPT, 1 μM, 1 h followed by 1 h recovery) was used as a positive control for ssDNA formation induced by DSBs. Data are shown as representative immunofluorescent microscopy images of BrdU foci (ssDNA) in S-phase (CldU positive) HeLa cells (d), and as quantification of cells with more than 15 BrdU foci (e). Scale bar = 10 µm. >70 individual CldU positive cells were scored per condition per experiment. Mean ± SEM; n = 3 experimental replicates, two-tailed Student's t-test. f FA treatment and SPRTN-depletion fail to induce phosphorylation of CHK1 at Ser345 due to the absence of ssDNA formation. HU or CPT were used at the same conditions as in (d) as positive controls for ssDNA-induced CHK1 and CHK2 activation. Immunoblots of HeLa cells whole cell extracts shown here represent three experimental replicates. Source data for (a–c, e, f) are provided as a Source Data file