Fig 1: Nuclear localisation of L1CAM inhibits γ-H2AX foci resolution and DNA repair in HUVECs.a Immunofluorescence staining and quantification of γ-H2AX foci colocalized with L1-CT (upper left panel; magnification, ×400) and immunoblotting (lower left panel) and quantification (right panels) of full-length L1CAM, L1-CT fragments, and γ-H2AX at 48 h post-irradiation (10 Gy) in HUVECs with or without γ-secretase inhibitor L-685,458 (3 µM) treatment. For quantification of γ-H2AX foci colocalized with L1-CT, error bars represent mean ± SEM (p = 0.0017). For quantification of full-length L1CAM and L1-CT fragments, error bars represent mean ± SD (full-length L1CAM: (-) vs. IR p = 0.0011; IR vs. IR + L-685,458 p = 0.0233, L1-CT fragments: (-) vs. IR p = 0.0017; IR vs. IR + L-685,458 p = 0.0177). b, c HUVECs were transfected with human full-length (L1-WT), NLS-mutated (L1-4A), and endocytosis-deficient (L1-dRSLE) L1CAM vectors after knockdown of endogenous L1CAM. b Scheme of L1-WT, L1-4A and L1-dRSLE constructs (top left panel). Immunoblotting of full-length L1CAM in HUVECs (top right panel) and immunofluorescence staining (middle panels) and quantification (bottom panels) of L1-CT and γ-H2AX in HUVECs 48 h post-irradiation (10 Gy; magnification, ×400). Scale bar = 5 µm. Error bars represent mean ± SEM (No. γ-H2AX foci: Control vs. L1CAM-WT p = 0.001; L1CAM-WT vs. L1CAM-4A p = 0.0001; L1CAM-WT vs. L1CAM-dRSLE p = 0.0002, No. L1-CT foci: ****p < 0.0001; L1CAM-WT vs. L1CAM-dRSLE p = 0.0004, No. colocalized foci: Control vs. L1CAM-WT p = 0.006; ****p < 0.0001). c Immunofluorescence staining and quantification of phalloidin and γ-H2AX in HUVECs 48 h post-irradiation (10 Gy; magnification, ×400). Scale bar = 20 µm. Error bars represent mean ± SD (Control vs. L1CAM-WT p = 0.0096; L1CAM-WT vs. L1CAM-4A p = 0.0014; ****p < 0.0001). d Immunofluorescence staining and pearson’s correlation coefficient of L1-CT colocalized with p-ATM, 53BP1, and DNA-PKcs in the nuclei of Ab417-pre-treated HUVECs 48 h post-irradiation (10 Gy magnification, ×400). Scale bar = 5 µm. Error bars represent mean ± SD (****p < 0.0001). e Flow cytometry analysis of cells with GFP positivity resulting from DNA repair and quantification of HR (upper panel) and NHEJ (lower panel) efficiency in L1CAM-knockdown HUVECs. The HUVECs were transiently transfected with the DR-GFP or EJ5-GFP construct along with control or L1CAM siRNA and were then transfected with a SceI plasmid to induce DNA damage. Error bars represent mean ± SD (HR efficacy p = 0.0005, NHEJ efficacy p = 0.0011). f Immunofluorescence staining (upper panel) for GFP, phalloidin, γ-H2AX in HUVECs 48 h post-irradiation (10 Gy). Quantification of phalloidin is shown (magnification, ×400; right panel). Scale bar = 20 µm. Error bars represent mean ± SD (No IR vs. IR + Control p = 0.0001; IR + Control vs. IR + L1CAM-WT p = 0.0063; IR + L1CAM-WT vs. IR + L1CAM-C-term p = 0.0027, IR + Control vs. IR + L1CAM-C-term p < 0.0001). HUVECs were transfected with L1-WT or L1-CT lentiviral vectors tagged with N-terminal GFP and C-terminal His. Transfection efficiency (left panel) was tested by immunoblotting for L1-WT and L1-CT. For quantification of foci colocalized with L1CAM, the colocalized foci in each sample were counted in a minimum of 70 cells per field (magnification, ×100). The average number of colocalized foci/cell was determined from five fields (magnification, ×100). The data are presented the means ± SDs and ±SEMs from three independent experiments. (a upper left panel: two-talied Student’s t-test; a all other panels and b, c, f: one-way ANOVA for multiple comparisons; d, e two-way ANOVA for multiple comparisons).