Journal: Communications Biology
Article Title: Condensin II interacts with BLM helicase in S phase to maintain genome stability
doi: 10.1038/s42003-025-07916-0
Figure Lengend Snippet: 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. S . 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.
Article Snippet: Commercially available antibodies against the following proteins are as follows with the application and concentration used: β-actin (Santa Cruz, cat. no. sc-69879, WB, 1:5000), Biotin (Jackson Labs, cat. no. 200-002-211, Immunofluorescence (IF), 1:1000; Bethyl, cat. no. A150-109A, IF, 1:1000), BLM (Santa Cruz, cat. no. sc-365753, WB and IF, 1:200 and 1:250; Bethyl, cat. no. A300-110A, IP, 5 μg/mL; Abcam, cat no. ab2179, IF, 1:2000), BrdU (BD Biosciences, cat. no. 347580, IF, 1:50; Abcam, cat. no. ab6326, IF, 1:100), CREST (Antibodies Inc., cat. no. 15-234-0001, IF, 1:50), Cyclin A (Santa Cruz cat. no. sc-239, WB, 1:500), Cyclin B1 (Santa Cruz cat. no. sc-245, WB, 1:1000), Cyclin E (Santa Cruz cat. no. sc-377100, WB, 1:500), GAL4-TA (Santa Cruz, cat. no. sc-1663, WB, 1:200), γ-H2AX (Abcam, cat. no. ab81299, WB, 1:5000), Histone H2A (Invitrogen, cat. no. MA5-24662, WB, 1:1000), Histone H4 (Santa Cruz, cat. no. sc-25260, WB, 1:200), MCM6 (Santa Cruz, cat. no. sc-393618, WB, 1:200), NCAPD3 (Santa Cruz, cat. no. sc-81597, WB, 1:200), NCAPG (Santa Cruz, cat. no. sc-515297, WB, 1:200), NCAPH (Santa Cruz, cat. no. sc-101013, WB, 1:500; Bethyl cat. no. A300-603A, IP, 5 μg/mL), NCAPH2 (Santa Cruz, cat. no. sc-393333, WB and IF, 1:500 and 1:250; Bethyl cat. no. A302-275A, IP, 5 μg/mL), PCNA (Cell Signaling, cat. no. 2586, WB, 1:5000), p-DRP1 (Cell Signaling, cat. no. 4494S, WB, 1:500), p-Histone H3 (Cell Signaling, cat. no. 9701S, WB, 1:500), RAD51 (Santa Cruz, cat. no. sc-398587, WB, 1:200), RPA2 (Santa Cruz, cat. no. sc-56770, WB, 1:200), SMC2 (Bethyl, cat. no. A300-058A, IF and Immunoprecipitation (IP), 1:1000 and 5 μg/mL; Invitrogen, cat. no. GT4312, WB, 1:1000; Cell Signaling, cat. no. 5329, IP, 5 μg/mL; Abcam, cat. no. ab10412, IF, 1:250) SMC4 (Bethyl, cat. no. A300-063A, WB, 1:1000), TOP2A (Santa Cruz, cat. no. sc-365916, WB, 1:200), TOP3A (Abcam, cat. no. ab108493, WB, 1:1000).
Techniques: Immunofluorescence, Microscopy, Labeling, Comparison, DNA Labeling, Fluorescence, MANN-WHITNEY, Transfection, Staining