Fig. 5: Condensin II contributes to BLM localization to nascent DNA.

a Immunofluorescence microscopy images of endogenous BLM, NCAPH2, SMC2 and EdU-labeled nascent DNA in WT cells. Nucleus is outlined in merged image and box represents ROI for magnified image. Scale bars, 10 μM and 1 μM (magnified image). Quantification of BLM, NCAPH2, and SMC2 colocalization with EdU in WT cells. Colocalization percentage was determined by counting the number of BLM, NCAPH2, or SMC2 foci that overlapped with EdU in comparison to the total number of BLM, NCAPH2, or SMC2 foci in an ROI for each nucleus. 20 nuclei were analyzed for each protein and median percentage of colocalization is reported. b SIRF assay of SMC2 in BLM-COMP, BLM-WMF^mut #1, and BLM-KO cells. Following nascent DNA labeling with EdU and EdU:biotin co-Click with biotin azide and Alexa Fluor 488 azide, PLA was performed using antibodies against biotin and endogenous SMC2. Nucleus is outlined in the merged image. Scale bars, 10 μM. Corrected total Cellular Fluorescence (ctCF) of the SMC2 SIRF signal/EdU ctCF is shown for individual nuclei (BLM-COMP, n = 150 nuclei; BLM-WMF^mut #1, n = 156 nuclei; BLM-KO, n = 79 nuclei) and the median presented by a horizontal line. Significance of differences between cell lines was determined using a Mann-Whitney test (ns, not significant). c SIRF assay of NCAPH2 in BLM-COMP, BLM-WMF^mut #1, and BLM-KO cells. Following nascent DNA labeling with EdU and EdU:biotin co-Click with biotin azide and Alexa Fluor 488 azide, PLA was performed using antibodies against biotin and endogenous NCAPH2. Nucleus is outlined in the merged image. Scale bars, 10 μM. Corrected total Cellular Fluorescence (ctCF) of the NCAPH2 SIRF signal/EdU ctCF is shown for individual nuclei (BLM-COMP, n = 99 nuclei; BLM-WMF^mut #1, n = 85 nuclei; BLM-KO, n = 85 nuclei) and the median presented by a horizontal line. Significance of differences between cell lines was determined using a Mann-Whitney test (ns, not significant). d SIRF assay of BLM in BLM-COMP, BLM-WMF^mut #1, and BLM-KO cells. Following nascent DNA labeling with EdU and EdU:biotin co-Click with biotin azide and Alexa Fluor 488 azide, PLA was performed using antibodies against biotin and endogenous BLM. Nucleus is outlined in the merged image. Scale bars, 10 μM. Corrected total Cellular Fluorescence (ctCF) of the BLM SIRF signal/EdU ctCF is shown for individual nuclei (BLM-COMP, n = 120 nuclei; BLM-WMF^mut #1, n = 125 nuclei; BLM-KO, n = 140 nuclei) and the median presented by a horizontal line. Significance of differences between cell lines was determined using a Mann–Whitney test and is reported as ****p ≤ 0.0001. e BLM SIRF assay in the BLM-COMP and BLM-WMF^mut #1 cell lines following transfection with NCAPH2 siRNA or mock siRNA. Following nascent DNA labeling with EdU and EdU:biotin co-Click with biotin azide and Alexa Fluor 488 azide, PLA was performed using antibodies against biotin and endogenous BLM. Nucleus is outlined in the merged image. Scale bars, 10 μM. Corrected total Cellular Fluorescence (ctCF) of the BLM SIRF signal/EdU ctCF was calculated for each nucleus (BLM-COMP siNCAPH2, n = 102 nuclei; BLM-COMP siMock, n = 89 nuclei; BLM-WMF^mut #1 siMock n = 94 nuclei) with the horizontal line representing the median. Significance of differences was determined by a Mann-Whitney test and is reported as ****p ≤ 0.0001. For additional controls for Fig. 5b–e see supplementary Fig. S5b. f Immunofluorescence microscopy of calyculin-induced premature chromosome condensation (PCC) in BLM-COMP, BLM-KO, and BLM-WMF^mut #1 cells. Following nascent DNA labeling with EdU and chromosome spreading, EdU:biotin Click-iT was performed with biotin azide and slides were hybridized with biotin antibody and stained with DAPI. Box represents ROI for magnified image. Scale bars, 10 μM. The chromosomes of three spreads from a single experiment for each cell line were analyzed to quantify the percentage of resolved and unresolved sister chromatids.