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. 2017 Nov 6;6:e30523. doi: 10.7554/eLife.30523

Figure 6. PLK1 controls the switching between the 53BP1-mediated cleavage free pathway to BRCA1-mediated cleavage pathway.

(A) Immunoblot showing the immunoprecipitation from extracts of HEK293 cells tranfected with control vector (Con) or vector expressing Flag-tagged PLK1. (B, C) Immunoblotting (B) and its quantification (C) showing PLK1 levels after replication inhibition. The mean and s.d. from three independent experiments are shown. (D) QIBC analysis of PLK1 expression after replication inhibition. Asynchronous HCT116 cells were treated with 2 mM HU as indicated time before fixing. PLK1 levels of S phase (Figure 6—figure supplement 1A) were gated and plotted. Red lines indicate the medians of PLK1. (E) Immunoblot showing the immunoprecipitation of FLAG-tagged MUS81. Suspension HEK293 cells expressing FLAG-tagged MUS81 were treated with or without nocodazole (100 ng/ml) and PLK1 inhibitors (10 μM BI2536 or BI6727) for 17 hr and 5 hr before harvest, respectively. (F) QIBC analysis of wild-type and 53BP1-/- HCT116 cells treated with 2 mM HU and PLK1 inhibitor (10 μM BI2536) for 3 hr. ****p<0.0001. (G) DNA combing assay showing that PLK1 works in the same pathway with BRCA1 in stalled fork restart. The sketch above delineates the experimental design. The mean and s.d. from three independent experiments are shown. **p<0.01, ns p>0.05. Please refer to Figure 6—figure supplement 1 for additional information in support of Figure 6.

Figure 6.

Figure 6—figure supplement 1. PLK1 promotes the recruitment of MUS81 to stalled replication forks.

Figure 6—figure supplement 1.

(A) QIBC analysis of PLK1 expression after replication inhibition. Asynchronous HCT116 cells were treated with 2 mM HU as indicated time before fixing. The mean levels of PLK1 in the S and G1 phase were showed by red dash lines and green dash lines, respectively. (B) Immunoblot showing the immunoprecipitation of FLAG-tagged SLX4. Suspension HEK293 cells expressing FLAG-tagged SLX4 were treated with or without nocodazole (100 ng/ml) and PLK1 inhibitors (10 μM BI2536 or BI6727) for 17 hr and 5 hr before harvest, respectively. (C) Immunoblot showing protein levels of BRCA1, BARD1, SLX4 and MUS81 on the chromosome. Asynchronous cells were treated with or without 10 μM BI2536 and 5 mM HU for 16 hr before harvest. (D–E) Immunofluorescence (D) and its quantifications (E) showing colocalization of MUS81 and FANCD2 in condensed mitotic nuclei. Experiments were preformed as the workflow in Figure 6—figure supplement 1. PLK1 inhibitor (10 μM BI2536) was added after G2 phase release. (F–G) Immunofluorescence (F) and its quantifications (G) showing colocalization of BRCA1 and ssDNA. HeLa cells were labeled with BrdU for 24 hr and then treated with or without 2 mM HU and 10 μM BI2536 for 5 hr. The Pearson coefficient of colocalization between BRCA1 and BrdU was measured by Huygens Professional Analysis Software. The mean and s.d. from three independent experiments are shown. ****p<0.0001, ***p<0.001, ns p>0.05. Scale bar, 5 μm.