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. 2016 Jan 6;7(3):2269–2283. doi: 10.18632/oncotarget.6825

Figure 2. Inhibition of Plk1 impairs BRCA1 foci formation following DNA damage.

Figure 2

A. BRCA1 foci formation induced by DNA damaging treatment is impaired when Plk1 is inhibited. HeLa cells were pre-treated or not for 2 h with BI2536 and then treated with calicheamicin (CLM) for 1 h or left untreated, washed and collected at different time-points following treatment to perform immunofluorescence assay. Cells were immunostained with anti-BRCA1 and anti-Rad51 antibodies, probed with DAPI and then examined by confocal fluorescence microscopy. Representative images of BRCA1 (green) and Rad51 (red) co-staining in control cells (+ CLM) or in BI2536-pretreated cells (+ BI2536; + CLM) 2 h after CLM treatment are shown. B. The number of Rad51 foci-positive cells is reduced when Plk1 is inhibited. The number of foci was quantified using ImageJ software (NIH). Graph shows the mean number of positive cells containing more than 5 foci Rad51 foci ± SE over 3 independent experiments, n ≥ 120 cells per time-point. Significant differences in Rad51-positive cells numbers were assessed using a two-tailed unpaired Student's t-test and are indicated by *** = p < 0.001. C. The number of BRCA1 foci-positive cells is reduced when Plk1 is inhibited. The number of foci was quantified using ImageJ software (NIH). Graph shows the mean number of positive cells containing more than 5 foci BRCA1 foci ± SE over 3 independent experiments, n ≥ 150 cells per time-point. Significant differences in BRCA1-positive cells numbers were assessed using a two-tailed unpaired Student's t-test and are indicated by ** = p < 0.01 and *** = p < 0.001. D. Downregulation of Plk1 by siRNA was analyzed in HeLa cells. 24 h after transfection with 10 nM control siRNA (siCont) or siRNA against Plk1 (siPlk1), whole-cell extracts were resolved by SDS-PAGE and immunoblotted using anti-Plk1 antibody. Equal loading was confirmed using anti-Hsp60 antibody. E. The number of BRCA1 foci-positive cells is reduced when Plk1 is downregulated. BRCA1 foci were examined 6 h following CLM treatment. The number of foci was quantified using ImageJ software (NIH). Graph shows the mean number of positive cells containing more than 5 BRCA1 foci ± SE over 3 independent experiments, n ≥ 120 cells per time-point. Significant differences in BRCA1-positive cells numbers were assessed using a two-tailed unpaired Student's t-test and are indicated by *** = p < 0.001. F. The number of Rad51 foci-positive cells is reduced when Plk1 is downregulated. Rad51 foci were examined 6 h following CLM treatment. The number of foci was quantified using ImageJ software (NIH). Graph shows the mean number of positive cells containing more than 5 Rad51 foci ± SE over 3 independent experiments, n ≥ 120 cells per time-point. Significant differences in Rad51-positive cells numbers were assessed using a two-tailed unpaired Student's t-test and are indicated by ** = p < 0.01.