Fig 1: Biological penetrance of mitotic perturbation determines the timing and magnitude of mitosis-born DNA breakage.(a) A representative image (n=50) of U-2-OS cells treated with siRNA to CDC27 and immunostained for ?-H2AX. Nuclear DNA was counterstained with DAPI. Scale bar, 10 µm. The bar charts (left) show ?-H2AX-decorated DSBs in mitotic cells (n=250) in the indicated times after siRNA transfection. (b) U-2-OS cells were treated with siRNA to PLK1 and analysed as in a. Scale bar, 10 µm. (c) Representative image galleries (n=25) of U-2-OS cells expressing mRFP-53BP1, treated with the indicated siRNAs, and analysed by time-lapse microscopy. Average mitotic duration (n=25) for each siRNA treatment is indicated. Numbers indicate time (h) after siRNA transfection. Scale bars, 10 µm. (d) Representative image galleries (n=25) of U-2-OS cells expressing mRFP-53BP1, treated with the indicated siRNAs, and analysed as in c. Average mitotic duration (n=25) for each siRNA treatment is indicated. Numbers indicate time (h) after siRNA transfection. Scale bars, 10 µm.
Fig 2: Immunoblot analysis of miR-186 predicted targets CDC27 and BUB1 (Loading control: vinculin) in clones transduced with scrambled control or miR-186 overexpressing lentivirus and exposed to 0 or 100 nM iAs for 4 weeks. A) Western blot of protein lysates from each clone. B) Densitometric and statistical analysis for CDC27 expression levels. Two-way ANOVA (pmiR-186 <0.001) followed by Tukey’s multiple comparison test *p < 0.05, * *p < 0.007. C) Densitometric analysis for BUB1 expression levels (no statistical differences). The values for each protein of interest were normalized against vinculin. The mean expression of unexposed scrambled control transduced samples was considered to be 100 % and the expression of iAs-exposed scrambled control transduced, and miR-186 overexpressing clones (with or without iAs) were expressed as % mean control. Color scheme: scrambled control transduced are blue, miR-186 overexpression are pink; lighter blue and pink circles represent clones without iAs exposure, while darker blue and pink circles represent clones exposed to 100 nM iAs.
Supplier Page from Abcam for Anti-Cdc27 antibody [EPR7798]