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. 2015 Mar 26;10(12):2006–2018. doi: 10.1016/j.celrep.2015.02.059

Figure 3.

Figure 3

RB Family Proteins Promote PRKDC-Dependent NHEJ

(A and B) Reporter-based quantification of NHEJ repair. Cells were transfected with NHEJ reporter and I-SceI encoding plasmid to introduce DSBs, with repair by NHEJ yielding GFP-positive cells (A). DsRed was used to identify transfected cells. Assays were quantified using flow cytometry. Representative raw images for HCT116 cells treated with siRNA targeting RB1 or RB1, RBL1, and RBL2, or a non-targeting control (siNT) are shown (B) NHEJ repair proficiency is determined by calculating the ratio of GFP-positive (Q2) versus DsRed-positive cell (Q1 + Q2).

(C) Graph depicting repair proficiency in NHEJ reporter-transfected cells. Cells were treated as for (B). NHEJ repair proficiency for cells transfected with siNT was set to 1. Binding of XRCC5 by RBL1 and RBL2 shown in Figure S3.

(D) Cell-cycle profiles for DsRed-positive cells from (C).

(E) Effect of PRKDC inhibition on reporter repair proficiency. HCT116 cells were treated and evaluated as for (C). DMSO or the PRKDC inhibitor NU7441 (5 μM) was added for the duration of the experiment.

(F) Immunodetection documenting RB1 and RB protein family loss in HCT116. HCT116 transfected with siRNA as in (C)–(E). Lysates probed using antibodies as indicated.

(G) NHEJ proficiency in SAOS-2 (RB1-negative) and U2OS (RB1-positive) cells using transient NHEJ reporter transfection as for (C) and (D).

(H) Effect of PRKDC inhibition on NHEJ proficiency. SAOS-2 and U2OS cells with integrated NHEJ reporter were used. Lines were transfected with I-SceI and DsRed plasmids and treated with DMSO or NU7441 as for (E). For all experiments, the average for n = 3 biological replicates is depicted; error bars ±SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 using a paired Student’s t test.

(I) Immunodetection of PRKDC-Ser2056 phosphorylation status. Autophosphorylation of PRKDC-Ser2056 signifies PRKDC activity. Cells were treated with 5 μM NU7441 for 24 hr prior to 5 Gy IR. 2 hr post IR, the cells were harvested into SDS protein loading buffer. Actin and total PRKDC levels are shown as loading controls.