Fig 2: Status of RAD50, NPM1 and XRCC5 in DSB-repair pathway and modulation of their expression on E2 and cisplatin treatment. A. Schematic diagram showing designations of RAD50, NPM1 and XRCC5 in HR and NHEJ pathways of DSB-repair. B. Representative immunoblot showing elevated expression of c-Myc on 10nM E2 treatment (upper panel); fold-change expression of c-Myc, NPM1 and RAD50 mRNA and protein (lower panel). C. Representative immunoblots of H2AX-?, NPM1, RAD50 and XRCC5 on 4 µM Cisplatin treatment; numbers indicate mean fold-change values. D. Immunofluorescence staining indicating NPM1, RAD50 and XRCC5 expression on 4 µM Cisplatin treatment in comparison to untreated control A4-T cells; Scale bar 25 µm. E. Immunofluorescence staining showing translocation of NPM1 from nucleolus to nucleoplasm within 24 h upon 4 µM cisplatin treatment in A4-T cells in comparison to untreated cells. Scale bar 8 µm. Data shown as mean ± SE of triplicate experiments. *p <0.05,**p <0.01.

Fig 3: DSB response triggers p53 signaling under genomic stress. A. Immunofluorescence staining showing expression of RAD50, NPM1, XRCC5, H2AX-? and p53 at steady state and 4 µM cisplatin treated control siRNA, siRAD50, siNPM1 and siXRCC5 A4-T cells. B. Quantitation of ATM, H2AX-?, p53, p21, CDK1, Cyclin D1 and Bcl-2 protein expressions in untreated and cisplatin treated A4-T, control siRNA, siRAD50, siNPM1 and siXRCC5 A4-T cells. C. In-Situ fluorescein cell death (TUNEL) analysis showing nuclear fragmentation (red foci) in untreated and cisplatin treated control siRNA, siRAD50, siNPM1 and siXRCC5 A4-T cells (Red color- pseudo color instead of green). D. Quantitative analysis of TUNEL assay showing percentage apoptotic populations in complete set of untreated and cisplatin treated A4-T, control siRNA, siRAD50, siNPM1 and siXRCC5 cells. Scale bar is 12 µm. Data shown as mean ± SE of triplicate experiments. *p <0.05; **p <0.01; ***p <0.001.

Fig 4: α-LA inhibits H. pylori-induced decreases in the nuclear Ku70/80, Ku–DNA binding activity, and ubiquitination of Ku80 in AGS cells. The cells were incubated with α-LA at the indicated concentrations for 2 h, then stimulated with an H. pylori to AGS cells ratio of 100:1 for 24 h. (A) Western blots of Ku70/80 in nuclear extracts. Actin and lamin B1 served as the loading control and nuclear marker, respectively. (B) The activity of Ku binding to DNA in the nuclear extracts was determined by an electrophoretic mobility shift assay. (C) Whole-cell extracts were immunoprecipitated (IP) with the anti-ubiquitin antibody, followed by Western blot analysis (WB) with anti-Ku80 antibody. The levels of the input proteins, Ku80 and actin, were determined by Western blot analysis. Input was used as the control for the protein expression.

Fig 5: The catalytic activity of SIRT7 is required for efficient DSB repair.(a) NHEJ efficiency was determined by FACS in SIRT7- or Ku80-deficient EJ5-HEK293 cells. Each bar represents the mean±s.d. for triplicate experiments. KD efficiency of SIRT7 and Ku80 was examined by western blotting. (b) HR efficiency was determined by FACS in SIRT7- or BRCA1-deficient DR-GFP-U2OS cells. Each bar represents the mean±s.d. for triplicate experiments. KD efficiency of SIRT7 and BRCA1 was examined by western blotting. (c) Rescue experiments for NHEJ or HR deficiency induced by SIRT7 depletion. EJ5-GFP-HEK293 cells (left) or DR-GFP-U2OS cells (right) stably expressing SIRT7 siRNA-1-resistant SIRT7wt (rSIRT7wt) or siRNA-1-resistant SIRT7H187Y (rSIRT7H187Y) were transfected with control siRNA or siSIRT7-1 as indicated. Twenty four hours later, the cells were transfected with pcDNA3.1 vector or I-SceI for 48 h, and collected and analysed by FACS. Each bar represents the mean±s.d. for triplicate experiments. (d) SIRT7 occupancy at chromatin flanking DSB generated by endonuclease I-SceI. DR-GFP-U2OS cells transfected with I-SceI were collected at different time points and subjected to ChIP assay, with antibodies against SIRT7. The final DNA extractions were amplified by quantitative real-time PCR using primer that covers the DNA sequences flanking the I-SceI site. Each bar represents the mean±s.d. for triplicate experiments (left). Control or DR-GFP-U2OS cells stably expressing rSIRT7wt or rSIRT7H187Y were transfected with control siRNA or siSIRT7-1 as indicated. Twenty four hours later, the cells were transfected with pcDNA3.1 vector or I-SceI for 40 h and subjected to qChIP analysis with antibodies against SIRT7. Each bar represents the mean±s.d. for triplicate experiments (right). (e) The efficiency of SIRT7 KD and overexpression of I-SceI, rSIRT7wt or rSIRT7H187Y in DR-GFP-U2OS cells. Tubulin was analysed as an internal control. (f) The KD specificity of SIRT7 siRNA-1 for FLAG-tagged wild-type SIRT7, siRNA-1-resistant rSIRT7wt or rSIRT7H187Y in DR-GFP-U2OS cells. The haemagglutinin (HA)-tagged I-SceI and tubulin were used as loading controls. **P<0.01 (two-tailed unpaired Student's t-test).