Fig 2: Tax expression switch DDSB repair from HR to error-prone NHEJ.A) Colocalization of Ku80 (specific to NHEJ DNA repair) and ?-H2AX in MT-2 and WT4 cells 30 min after gamma-irradiation. B) Colocalization of ?H2AX foci and Ku80 in Tax-inducible JPX9 after induction of Tax expression by CdCl. Non-induced JPX9 and CdCl treated Jurkat control cells were used to demonstrate specificity to Tax expression. The images were obtained using epifluorescence Nikon Ti-s and have been treated with the deconvolution feature provided with Nikon's NIS-Elements software. C) Colocalization of ?-H2AX foci and Rad51 (specific to HR DNA repair) after Tax induction in JPX9 and in Jurkat cells that were used as control. The images were obtained using epifluorescence Nikon Ti-s and have been treated with the deconvolution feature provided with Nikon's NIS-Elements software.

Fig 3: The combined use of ZSTK474 and TMZ reduced HR repair efficiency. (a)–(d) SF295 and U87 cells were treated with ZSTK474 and TMZ as single agents or in combination for 48 h. (a) The formation of RAD51 foci was assessed using immunofluorescence. Cell nuclei were stained with DAPI. Scale bar, 20 µm. Representative images are shown. (b) Quantification of the number of RAD51-positive SF295 and U87 cells. (c) The cells were collected for western blotting detection of NBS1, p-ATM, BRCA1, BRCA2, DNA-PKcs, and Ku80. (d) Western blotting analysis of PI3K-p110ß, p-Akt, p-mTOR, and c-Myc. All data are presented as the mean ± SD (n = 3). *P < 0.05; **P < 0.01.

Fig 4: CYREN interaction with Ku in S and G2 inhibits cNHEJ.a, Immunoblotting of a peptide binding array of full length CYREN-1. Each dot represents 20 aa of CYREN-1 with 19 aa overlap with the previous and following peptide. Upper panel, ponceau. Middle panels, duplicate incubation with Ku70/Ku80 recombinant proteins and immunoblotting with anti-Ku70 antibody. Lower panel, control immunoblotting with anti-Ku70 antibody without incubation with recombinant Ku70/80. For gel source data, see Supplementary Figure 1. b, Alanine scan of CYREN-1 on residues 9 to 46. FLAG immunoprecipitation of protein extracts from HEK293T cells transfected with pCDNA3.1 plasmids expressing wt CYREN-1-FLAG or each of the single residues mutated to Alanine. Total lysate and FLAG-IP were then immunoblotted with anti FLAG or Ku70 antibodies. For gel source data, see Supplementary Figure 1. c, Protein alignment of CYREN-1 Ku Binding Motif among vertebrates. d, Photo-crosslinked pull down of a BPA-BIO-CYREN(2-24) peptide in HEK293T protein extract, followed by immunoblotting with anti-Ku70 and Ku80 antibodies. + and ++, 25µM and 50µM of BPA-Bio-CYREN. 100µM of CYREN(2-24) free peptide was used as a competitor. For gel source data, see Supplementary Figure 1. e, Experimental outline of Fig. 4d. HT1080 6TG cells stably expressing an inducible control GFP or wt or mutant CYREN-1-3xFlag were transduced with shTRF2 on Day 0 and transfected with a control (NT) pool of siRNAs or a pool of siRNAs targeting the 3’UTR of CYREN on Day 2. Expression of CYREN-1 wt and mutants was induced on Day 3 and cells were collected on Day 5. f, FLAG western blot of endogenous C-terminal 3FLAG tagged CYREN-1 and CYREN-2 cells following double thymidine synchronisation. For gel source data, see Supplementary Figure 1.

Fig 5: Mdm2 expression is strongly impacted by IAV infection, mostly at post-transcriptional levels. (A) Human lung A549 cells were mock-infected or infected by influenza A/Moscow/10/99 (H3N2) at a MOI of 0.1 and cell lysates were analyzed by western blot for the expression of Mdm2 (SMP14 antibody), p53 and IAV NS1 at different time-points post infection. Ku80 was used as a loading control. Mdm2 relative protein levels (Mdm2 RPL) were measured by densitometry and calculated from data from three independent experiments. An asterisk indicates a significant difference compared with the results for mock-infected cells (** and *** for P-value < 0.01 and 0.001, respectively). (B) Alternatively, A549 or H1299 cells were mock-infected or infected by influenza A/Moscow/10/99 (H3N2) at different MOI and expression of Mdm2, p53 and IAV NS1 were monitored at 24 h post-infection by western blot, using the same approach. (C) A549 cells were mock-infected or infected by influenza A/Moscow/10/99 (H3N2) at a MOI of 4 or 0.1 and cell supernatants and lysates were harvested at 4, 8 or 24 hpi, respectively. Endogenous Mdm2 expression was measured at protein level by western blot, Mdm2 promoter activity was monitored using a luciferase reporter plasmid. In addition, Mdm2 mRNA expression was measured by RT-qPCR. An asterisk indicates a significant difference compared with the results for mock-infected cells (*, **, *** for P-value < 0.05, 0.01 and < 0.001, respectively).