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. Author manuscript; available in PMC: 2018 Oct 19.
Published in final edited form as: Mol Cell. 2017 Oct 19;68(2):414–430.e8. doi: 10.1016/j.molcel.2017.09.036

Figure 1. Analysis of nascent DNA degradation in BRCA1/2- and FANCD2-deficient cells upon SMARCAL1 depletion.

Figure 1

(A) Detection by western blot of BRCA1 (left), BRCA2 (right) and SMARCAL1 protein levels in MCF10A cells subjected to shRNA-dependent depletion. β-actin levels are shown as loading controls.

(B) Schematic of CldU/IdU pulse-labeling followed by a 5 hr hydroxyurea (HU; 2 mM) treatment (top). Representative images of CldU and IdU replication tracks in HU-treated MCF10A cells expressing the indicated shRNAs (bottom).

(C) Dot plot of IdU to CldU tract length ratios for individual replication forks in HU-treated MCF10A cells expressing the indicated shRNAs with or without mirin (50 μM). The median value of 200 or more IdU and CldU tracts per experimental condition is indicated. Statistical analysis was conducted using Mann-Whitney test (n.s. not significant, **** p<0.0001). Data are representative of 3 independent experiments.

(D) Schematic of CldU/IdU pulse-labeling followed by HU treatment as in (B) (top) and dot plot of IdU to CldU tract length ratios for individual replication forks in HU-treated PD20 cells with or without complementation with FANCD2 cDNA and treatment with SMARCAL1 siRNA (bottom). Data are shown and analyzed as in (C) and represent 2 independent experiments.

(E) Schematic of the CldU/IdU pulse-labeling assay (top) and dot plot of IdU to CldU tract length ratios for individual replication forks in MCF10A cells with or without combined treatment with HU (2 mM) and B02 (25 μM) for 5 hrs (bottom). Data are shown and analyzed as in (C) and represent 2 independent replicates.

(F) Schematic of the CldU/IdU pulse-labeling assay conducted as in (B) (top) in control and SMARCAL1 KO MCF10A cells subjected to control or BRCA1 siRNA treatment with or without expression of wild-type (WT), R764Q- or ΔN1-115-mutant SMARCAL1 proteins. Representative images of CldU (red) and IdU (green) replication tracks in the MCF10A cells indicated above after HU treatment (bottom).

(G) Dot plot of IdU to CldU tract length ratios for individual replication forks in the MCF10A cells shown in (F) after HU treatment. Data are shown and analyzed as in (C) and represent 2 independent experiments. See also Figure S1.