Fig 2: Two essential, limiting DNA replication factors are regulated at the G1/S transition through the destruction of TICRR/TRESLIN by the CRL4DTL E3 ubiquitin ligase.

Fig 3: Overexpression of Treslin in Ensa siRNA-treated cells rescues normal S phase. a Parental or U2OS cells stably overexpressing AcGFP-Flag-Treslin were transfected with scramble (siSC) or Ensa siRNAs #1 (siE1) or #2 (siE2) (for parental) or with Ensa siRNA #1 and #2 (for AcGFP-Flag-Treslin overexpressing cells) and the levels of Ensa and ß-tubulin as a loading control, checked by western blot. Cells were synchronised in G1–S by thymidine block 24 h later and recovered for western blot at 7.5 h b and 10 h c after release. The levels of Treslin in non-transfected U2OS cells stably overexpressing AcGFP-Flag-Treslin were also checked (CT). FACS analysis at 7.5 h d and 10 h e after release are also shown

Fig 4: Ensa KD increases S-phase length by decreasing fork number without affecting origin licensing. a Diagram depicting IdU (red)–CldU (green) double pulse analysis by DNA combing. b Representative image of a triple-stained DNA fibre: IdU (red)–CldU (green)–DNA (blue). c Fork velocity (Kb/min) and d global instant fork density (GIFD) values (fork per Mb) are represented. Mean ± standard deviation and p-value obtained by using two-tailed unpaired Student’s t-tests, are represented. Data pooled from four biological replicates. e Diagram representing the experiment in which HeLa cells were blocked in G1–S phase by thymidine for 24 h and subsequently released with fresh medium containing nocodazole (50 ng/ml) for a supplementary period of 16 h before shakeoff. Four hours later, released cells were transfected with siSC or siEnsa, and after 1 h, supplemented with fresh medium containing thymidine for an additional 18 h incubation. Cells were then released and recovered at the indicated times for FACS analysis. f FACS profiles of SC (red) and Ensa (blue) siRNA-treated cells at different time points after release are shown. g HeLa and U2OS cells were transfected with scramble (siSC) or Ensa (siEnsa) siRNAs and 48 h later processed for chromatin isolation as reported in Methods section. Cytosolic (S2) and nuclear (S3) soluble fractions as well as chromatin fraction (P3) were then used for western blot analysis to determine Treslin and MCM 2, 3 and 4 levels. h As for g, except that MCM 5 and 7, histone H3 and ß-tubulin levels were checked. IOD, inter-origin distances; Ori, replication origin

Fig 5: DTL knockdown increases TICRR protein levels in S phase. (A) TICRR-mClover signal, measured as in Figure 3A, 24 or 48 h after siRNA transfection. (B) Quantification of capillary electrophoresis of whole cell lysates prepared 24 h after siDTL-pool transfection probed for DTL/CDT2 (anti-CDT2) normalized to Lamin (anti-Lamin) (n = 3). P-value is from Student's t-test (*P< 0.05) (C) Capillary electrophoresis of whole cell lysates 24 h after siDTL-pool transfection probed for TICRR (anti-GFP) and Lamin (anti-Lamin). (D) Immuno-flow cytometry measurement of total TICRR-mClover (anti-GFP) and DNA content (propidium iodide) 24 h after siDTL-pool transfection. Black line represents upper limit of background signal from negative control. (E) Quantification of median TICRR-mClover signal in 2N, early-S, and 4N cells from D (n = 3 independent experiments). Values are normalized to untreated cells. Midlines and error bars mark the mean ± SD. P-values are from two-way ANOVA with Dunnett's Post Hoc Test (*P< 0.05). (F) EdU incorporation and DNA content (propidium iodide) were measured by flow cytometry 24 h after siDTL-pool transfection. Gray box represents EdU+ cells. Horizonal lines represent median intensity of G1 (bottom) and EdU+ (top) cell populations. (G) Median EdU signal intensity was normalized to that of untreated cells. P-value is from Student's t-test, ns.