Fig 1.
Synthetic lethality of DDX11 and ESCO2 is conserved in fibroblasts of WABS and RBS patients.
(A) WABS fibroblasts and corrected cells were transfected with the indicated siRNAs and cell viability was analyzed after four days, using a cell-titer blue assay. (B) Cells were transfected with indicated siRNAs and analyzed by western blot after three days. WCE, whole cell extract. (C) Cohesion defects were quantified in metaphases spreads of cells transfected as in B. Examples of metaphase chromosomes with normal and railroad (RR) appearance, as well as premature chromatid separation (PCS), are shown. (D, E) RBS fibroblasts and corrected cells were transfected with the indicated siRNAs and assessed after three days by cohesion defect analysis and western blot.
Fig 2.
Synthetic lethality of DDX11 and ESCO2 in RPE1 cells.
(A) CRISPR-Cas9 was used to knockout TP53 and DDX11 in RPE1-hTERT cells. The resulting isogenic cell lines were transfected with indicated siRNAs and proliferation was assessed using the IncuCyte. (B) Cells were transfected as in A and analyzed for cohesion defects. (C) CRISPR-Cas9 was used to disrupt the ESCO2 gene in RPE1-hTERT-TP53KO cells. Cells were transfected with indicated siRNAs and analyzed for cohesion defects.
Fig 3.
Induction of 4N fractions upon ESCO2 knockdown in WABS fibroblasts.
WABS cells were transfected with the siESCO2, harvested at the indicated time points and analyzed by western blot (A) and flow cytometry (B). (C) Quantification of two independent experiments.
Fig 4.
Restoring arm cohesion by WAPL knockdown rescues PCS and lethality.
(A,B) RBS cells were transfected with siWAPL and harvested after three days for western blot and analysis of cohesion defects. (C-E) WABS cells were transfected with the indicated siRNAs and harvested after three days for western blot and analysis of SCC. Viability was assessed after four days with a cell-titer blue assay.
Fig 5.
ESCO1, ESCO2 and DDX11 have separable functions in SCC.
(A,B) RBS fibroblasts and corrected cells were transfected with siRNA targeting ESCO1 and harvested after three days for Western blot and SCC analysis. Mean and standard deviations of two technical replicates are shown. (C,D) RBS cells were transduced with lentiviral vectors expressing the indicated proteins and selected with puromycin. Overexpression was confirmed with qRT-PCR and cells were analyzed for cohesion defects. Mean and standard deviations of two independent experiments are shown. Fold changes were calculated relative to parental cells. Note that mouse DDX11 mRNA is not detected in human cells, so these values represent arbitrary units (*). DDX11 (E,F) WABS cells were transduced with lentiviral vectors expressing the indicated proteins and selected with puromycin. Overexpression was confirmed with qRT-PCR and cells were analyzed for cohesion defects. Mean and standard deviations of two independent experiments are shown. Fold changes were calculated relative to parental cells.
Fig 6.
DDX11-Q23A mutant fails to rescue sister chromatid cohesion.
(A) WABS fibroblasts were stably transfected with WT-DDX11 or the DNA binding mutant DDX11-Q23A, and assessed for protein levels using western blot. Lines within blots indicate positions where irrelevant lanes were removed. (B) The expression of ectopic DDX11 was assessed by immunofluorescence. (C) Cells were analyzed for cohesion defects. Mean and standard deviations of two independent experiments are shown.
Fig 7.
DDX11 deficiency slows replication fork speed and reduces SMC3 acetylation.
(A) RPE1-hTERT cells were transfected with indicated siRNAs for two days and analyzed by western blot. Replication fork speed was assessed with a DNA fiber assay using a double labeling protocol. Black lines indicate the median. P-values were calculated by a non-parametric one-way ANOVA test. **** p<0,0001. Representative images of DNA fiber tracts are shown. (B) RPE1-TP53KO and RPE1-TP53KO-DDX1KO cells were analyzed with a DNA fiber assay. (C) WABS fibroblasts and corrected cells were transfected with the indicated siRNAs and analyzed by western blot after three days. Additional antibody incubations are provided in S2 Fig. (D) Quantification of AcSMC3 levels normalized to total SMC3, using image-lab software and. Mean and standard deviations are shown. P-values were calculated using a two-tailed t-test. * p<0,05; **<0,01; ns not significant. (E) RPE1-TP53KO and RPE1-TP53KO-DDX11KO cells were arrested in G1 using the Cdk4/6 inhibitor Palbociclib (20h, 10 μM) and in G2 using the Cdk1 inhibitor RO3306 (20h, 10 μM) and assessed by flow cytometry. (F) cells from (E) were lysed, and soluble and DNA-bound protein fractions were separated and analyzed by western blot. Ctrl, TP53KO cells; DX, TP53KO-DDX11KO cells.