Two independent experiments were performed. NSMCE2 null cells were pulsed for 40 min to account for the slower cell cycle.(TIF) pgen.1007942.s001.tif (2.3M) GUID:?E3390FDF-804F-4E8B-8C7A-9D173C44E191 S2 Fig: (A) Representative Western blots of HeLa cells transfected with control or two different siRNAs against NSMCE2 and treated or not with 2 mM HU for 24 hours. Multiple loading controls (HSP90) are shown for individual gel runs and Westerns of the same cell lysate. (B) Western blot analysis of SMC5. For SMC5 experiments, -actin was used as a loading control.(TIF) pgen.1007942.s002.tif (3.3M) GUID:?A08E248D-5566-4C48-98AD-6E45C8695596 S3 Fig: (A) Complementation of accumulation of BLM foci by transfection of siRNA-resistant NSMCE2 cDNA construct. HeLa cells were exposed to control or NSMCE2 siRNAs and were treated with 2 mM HU for 24 hours. Box and whisker plots represent distributions of the number of BLM foci per cell. The median values are shown in boxes. At least 10,000 BLM foci were analyzed in each experimental condition. Three impartial experiments were performed. (B) A representative image of the colocalization of RPA (red) and RAD51 (green) in HeLa cells exposed to 2 mM HU for 24 hours prior to fixation VX-702 (upper panel). Quantitation of the area of RAD51 foci (lower panel). Mean and standard error are shown. At least 10,000 RAD51 foci were analyzed in each experimental condition. Three impartial experiments were performed. (C) Colocalization of RAD51 and EdU in HU-treated cells. Representative VX-702 images of control and NSMCE2-depleted HeLa cells exposed to 2 mM HU for 24 hours. EdU VX-702 was incorporated for 12 min prior to HU treatment. After HU, cells were fixed and stained with RAD51. Images show the merge of EdU (green) and RAD51 (red) channels. (D) Reduced accumulation of RPA foci in HU-treated, NSMCE2-deficient U2OS cells. Box and whiskers plot represent distributions of the number of RPA foci in cells exposed to control or NSMCE2 siRNA and treated or not with 2 mM HU for 24 hours. The median values are shown in boxes. Three independent experiments were performed. (E) Complementation of accumulation of RPA foci by transfection of siRNA-resistant NSMCE2 cDNA construct. HeLa cells were exposed to control or NSMCE2 siRNAs and treated with 2 mM HU for 24 hours. Box and whiskers plot represent the distributions of the number of RPA foci per cell. The median values are shown in boxes. Three independent experiments were performed. (F) Reduced accumulation of chromatin-bound RPA in HU-treated NSMCE2 null cells compared to HU-treated normal HEK293T cells. Western blot analysis of levels of chromatin-bound RPA (RPA p70 subunit). Cells were treated or not with 2 mM HU for 16 hours. The M fraction contains equal parts of ADRBK1 the cytoplasmic and nucleoplasmic fractions. The C fraction contains the chromatin-bound material. The red carets point to the HU-induced chromatin-bound RPA. Four impartial experiments were performed. (G) Quantitation of the experiment shown in F. (H) Reduced levels of ssDNA in HU-treated NSMCE2-deficient cells. Quantitation of immunofluorescence analysis of BrdU to measure uncovered ssDNA in non-denaturing conditions (left panel). HeLa cells were exposed to control or NSMCE2 siRNAs and treated or not with 2 mM HU for 24 hours. The bar represents median values of the numbers of BrdU foci and the error bar represent the SEM values from three impartial experiments. Representative VX-702 images of BrdU foci are shown (right panel). (I) Comparable levels of SCEs in VX-702 normal HEK293T cells and NSMCE2 null cells. Box and whiskers plots represent the numbers of SCEs per metaphase. A minimum of 14 metaphases were scored in two impartial experiments. (J) Reduced levels of -H2AX in HU-treated, NSMCE2-deficient cells. Flow cytometric analysis of -H2AX response in HeLa cells. Mean and standard deviation is shown. To the right of the bar graph are representative histograms showing -H2AX induction. Shaded histograms represent the treated cell populations. Three impartial experiments were performed. (K) Complementation.