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. 2017 Oct 6;8(55):93319–93337. doi: 10.18632/oncotarget.21555

Figure 2. PRPF8 depletion and PlaB treatment cause a reduction end resection as detected by damage-induced chromatin bound RPA.

Figure 2

A. Cells were transfected with the siRNAs shown and cultured for 3 days prior to treatment with CPT (1 hr). For the small molecule treatments, cells were treated with siCTRL for consistency, and pre-treated with PlaB or DRB for 7 hours, prior to treatment with these small molecules and CPT (1hr). Cells were exposed to mild detergent extraction prior to fixation and staining with RPA34 or γH2AX, with DAPI counterstain. Shown are representative flow cytometry plots, as well as the percentage of cells showing detergent resistant (i.e., chromatin bound) RPA34 or γH2AX staining. *distinct from siCTRL/DMSO w/ CPT, P ≤ 0.0004. Targeting siRNAs N = 3, PlaB N = 5, DRB N = 4, siCTRL/DMSO w/ and w/o CPT N = 8 (higher N because a siCTRL/DMSO control was included each set of experiments). B. The treatments shown in A do not cause an increase in G1 phase cells. Cells were treated with siRNA as in A, and PlaB or DRB for 7.5 hrs (to maintain a total 8 hr treatment), followed by 30 minutes of pulse labeling with BrdU. Cells were stained for BrdU and counter stained with PI to determine the percentage of cells in G1, S, or G2, as shown. Shown are the mean values of N = 4 treatments, except siCTRL N = 8.