Fig 2: The synergistic anti-tumor effect of M3814 and etoposide in NSCLC xenografts. NOD-SCID mice implanted with A549 and H460 tumor xenografts were treated with M3814 (50 mg/kg, i.g. daily) and etoposide (10 mg/kg, i.p. once 3 days). (A) Subcutaneous A549 and H460 tumors at the end of this experiment. (B) and (C) Growth of A549 and H460 xenografts presented as mean RTV (relative tumor volume). Error bars denote SD. (D) and (E) The final RTV of xenograft tumors measured in each group. Data are presented as mean ± SD (n = 7, ****P < 0.0001, ***P < 0.001, *P < 0.05; ns, P > 0.05). (F) Representative images of immunohistochemistry staining of Ki67, ?-H2AX, pDNA-PKcs and immunofluorescence staining of TUNEL evaluated on paraffin-embedded A549 tumor sections, scale bar = 50 µm (magnification, 200 ×). Representative images of SA-ßGal staining of frozen sections of A549 tumors, scale bar = 50 µm (magnification, 200 ×).

Fig 3: WASH is required for DNA relaxation(A) WASHWT and WASHKO 3T3 cells were treated with 50 µM etoposide for the indicated time. Whole-cell lysates were obtained and immunoblotted with the indicated antibodies. GAPDH serves as a loading control.(B) Representative phosphorylation of DNA-PKcs (S2056) of shControl and shWASHa HeLa cells after etoposide treatment. HeLa cells stably expressing lentiviral shRNA against WASH or control shRNA were treated with 50 µM etoposide for 0, 15, 30, or 60 min. Then fluorescence intensity of p-DNA-PKcs (S2056) was assessed. Scale bar: 50 µm.(C) Expression levels of WASH in shControl and shWASHa HeLa cells were examined by immunoblot.(D) Fluorescence intensity of p-DNA-PKcs (S2056) per cell for 169–247 cells of each group were calculated and presented as mean ± SEM. Unpaired t test was performed between shControl and shWASHa. ****, p < 0.0001; n.s., not significant.(E) Chromatin relaxation induced by etoposide is lessened in the WASHKO cells. 3T3 WASHWT and WASHKO cells were treated with 50 µM etoposide for 60 min. Accessibility of chromatin was then measured by MNase digestion assay.(F) Quantified signal of the various gel lanes presented in (E).

Fig 4: WASH interacts with Ku protein(A) HeLa cells were transfected with HA-YFP.WASH and HA-YFP-NLS.WASH for YFP fluorescence imaging. Scale bar: 20 µm.(B) The nuclear proteins were extracted from HEK-293T cells expressing HA-YFP-NLS.WASH, subjected to subsequent anti-HA affinity purification, and analyzed by mass spectrometry. NHEJ factors associated with WASH identified by mass spectrometry are shown.(C) The interactions between endogenous WASH and Ku70/Ku80/DNA-PKcs were determined by co-immunoprecipitation (Co-IP) assay. HEK-293T cell lysates were immunoprecipitated in the absence or presence of EtBr (0.2 mg/mL) with normal rabbit serum (NRS) or anti-WASH followed by SDS-PAGE and immunoblotting.(D) The interactions between WASH and Ku70/Ku80 were confirmed by Co-IP assay. HEK-293T cells expressing the HA-YFP, HA-YFP.WASH, or HA-YFP-NLS.WASH were lysed and subjected to subsequent anti-HA co-immunoprecipitation followed by SDS-PAGE and immunoblotting.(E) The interactions between WASH and Ku70/Ku80 were assessed by PLA with the indicated pairs of primary antibodies in HeLa cells with 50 µM etoposide treatment for 2 h or not. Co-staining WASH with mouse IgG was used as negative control. Scale bar: 20 µm.(F) PLA signal foci numbers per nucleus for 81–123 cells of each group were calculated and presented as mean ± SD. Unpaired t test was performed between etoposide-treated and untreated groups. Two-way analysis of variance (ANOVA) with Dunnett's multiple comparisons test was performed among mIgG, Ku70, and Ku80 groups. ****, p < 0.0001; n.s., not significant. See also Figures S1 and S2.

Fig 5: Relationship between the DNA-PKcs expression and the recurrence-free survival in HCC patients undergoing liver resection.