Fig. 5.

Role of XRCC4 family members in the recruitment of Pol λ to laser microirradiation-induced DNA damage sites. a Upper, Schematic figure showing N-terminal EGFP- and mCherry-Pol λ fusion proteins; Lower, Representative immunofluorescence images showing that N-terminal EGFP- and mCherry-Pol λ fusion proteins are localised to nuclei in U2OS cells. b Recruitment of N-terminal EGFP-Pol λ to laser-induced DNA damage sites in U2OS cells. c Time course of N-EGFP-Pol λ recruitment to laser-induced DNA damage sites in U2OS cells. Data shown are the mean and SEM from 16 individual cells. d Immunoblot analysis of N-EGFP-Pol λ expressing U2OS cells deficient in PAXX, XLF or XRCC4 generated by CRISPR-Cas9. WCL were resolved by SDS-PAGE and the indicated proteins detected by immunoblotting. e Localisation of N-mCherry-Pol λ in U2OS WT, PAXX-, XLF- and XRCC4-deficient cells. Representative immunofluorescence images showing that N-terminal mCherry-Pol λ fusion protein localises to nuclei in PAXX-, XLF- or XRCC4-deficient U2OS cells. Cells were co-stained with DAPI or dynamin-2, a perinuclear-enriched protein. f Time course of N-EGFP-Pol λ recruitment to laser-induced DNA damage sites in U2OS-WT cells and cells deficient in PAXX, XLF or XRCC4. Data shown are the mean and SEM from WT, PAXX, XLF and XRCC4 knockout cells. Graphs shown are for the following cell numbers: WT: 8 cells; PAXX KO, 10 cells; XLF KO 17 cells; XRCC4 KO 18 cells. g Time course of N-EGFP-FLAG-Ku70 recruitment to laser-induced DNA damage sites in U2OS-WT cells and PAXX KO cells. Data shown are the mean and SEM from WT and PAXX knockout cells. Graphs shown are for the following cell numbers: WT: 17 cells; PAXX KO, 19 cells