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

Fig 3: PRPF8 forms nuclear structures that co-localize with XAB2, which are consistent with interchromatin granulesCells were treated with the siRNAs shown, followed by 10 Gy IR and 30 min recovery prior to mild detergent pre-extraction, fixation, and immunofluorescence analysis. Shown are immunofluorescence signals from representative cells (scale bar = 10 μm).

Fig 4: PlaB treatment causes PRPF8 to localize into mega-speckles, and disrupts XAB2 localizationCells were treated and analyzed as in Figure7, except including two different treatments of PlaB and DRB (8 hr and 18 hr total), during which the cells were also treated with 10 Gy IR treatment 30 minutes prior to pre-extraction and fixation. Shown are immunofluorescence signals from representative cells (scale bar = 10 μm).

Fig 5: Both PRPF8 depletion and PlaB treatment cause a reduction in H3K9Ac and H4K16Ac, and the latter causes a marked reduction in POL2-S2PCells were treated with siRNAs, PlaB, and DRB as in Figure 3, and chromatin was extracted for immunoblotting analysis of H3K9Ac, H4K16Ac, H3K9me3, POL2-S2P, and Actin. Shown are representative immunoblots from this analysis.