Fig. 1: L1-CT fragments colocalize with persistent γ-H2AX foci after irradiation (IR) or Dox treatment in HUVECs.

a, b Immunofluorescence staining and quantification of phalloidin, γ-H2AX and L1-CT 48 h post IR (2 Gy, 5 Gy, or 10 Gy) in HUVECs (left panels, magnification, ×400). Scale bar = 20 µm (enlarged, 5 µm). Bar graphs quantifying the number of γ-H2AX foci, the phalloidin density, and the number of colocalized foci (right panels). For quantification of phalloidin density, error bars represent mean ± SEM (2 Gy vs. 10 Gy p < 0.0001; 5 Gy vs. 10 Gy p = 0.0015). For quantification of the number of γ-H2AX foci, error bars represent mean ± SD (****p < 0.0001). Colocalized foci (marked by white arrow) are amplified and graphs represent quantification of L1-CT and γ-H2AX signals in selected regions of dotted lines (bottom panels). Error bars represented mean ± SEM (****p < 0.0001). c Heat map of the RNA-seq analysis results showing radiation-induced EndMT and the mesenchymal phenotype. Total RNA was isolated from HUVECs before and after 10 Gy IR (5 h, 72 h). d Immunofluorescence staining of L1-CT and γ-H2AX in HUVECs at 0, 1, 24, and 48 h post IR (10 Gy; left panels). Quantification of γ-H2AX foci and nuclear L1CAM (magnification, ×400; right panels). The number of γ-H2AX foci with an intensity greater than 40 and a foci diameter of 0.1 μm was counted. Scale bar = 20 µm (enlarged, 5 µm). e Immunoblotting and quantification of full-length L1CAM and L1-CT fragments from HUVECs transfected with lentiviral shRNA targeting L1CAM 48 h after IR (10 Gy; upper panels). Error bars represent mean ± SD (full-length L1CAM: sh-Control IR – vs. + p = 0.0003; sh-Control IR + vs. sh-L1CAM IR +  p < 0.0001, L1-CT fragments: sh-Control IR – vs. + p = 0.0013; sh-Control IR + vs. sh-L1CAM IR + p = 0.0006). Immunofluorescence staining of γ-H2AX and L1-CT 48 h post IR (10 Gy) in HUVECs (magnification, ×400). Scale bar = 5 µm. f Immunoblotting (upper panel) of full-length L1CAM and L1-CT fragments in the cytoplasmic (C) and nuclear (N) fractions of HUVECs 48 h post IR (10 Gy). Quantification of full-length L1CAM in the cytoplasmic fractions and L1-CT fragments in the nuclear fractions. HUVECs were treated with control IgG or Ab417 (20 µg/mL) before IR. GAPDH and lamin B were used as cytoplasmic and nuclear markers, respectively. Error bars represent mean ± SD from independent experiments (full-length L1CAM: No IR vs. IR + IgG p < 0.0001; IR + IgG vs. IR + Ab417 p = 0.0001, L1-CT fragments: No IR vs. IR + IgG p = 0.0228; IR +  IgG vs. IR + Ab417 p = 0.0448). g Immunofluorescence staining for L1-CT and γ-H2AX 0 and 48 h post IR (10 Gy) in HUVECs pre-treated with control IgG or Ab417 (20 µg/mL) (upper panel). Quantification of colocalization of γ-H2AX foci with L1CAM (magnification, ×400) (lower panel). Scale bar = 20 µm (enlarged, 5 µm). h Immunofluorescence staining (upper panel) and quantification (lower panel) of phalloidin and L1-CT 72 h post IR (10 Gy) in HUVECs pre-treated with control IgG or Ab417 (20 µg/mL; magnification, ×400). Scale bar = 20 µm. Error bars represent mean ± SD (p = 0.0007). i Immunofluorescence staining for L1-CT and γ-H2AX at 0 and 24 h after Dox treatment in HUVECs pre-treated with control IgG or Ab417 (20 µg/mL; left panel). Colocalized foci (marked by white arrow) are amplified and graphs represent quantification of L1-CT and γ-H2AX signals in selected regions of dotted lines (middle panels). Quantification of colocalization of γ-H2AX foci with L1CAM (magnification, ×400; right panel). Scale bar = 5 µm. j Immunofluorescence staining and quantification of phalloidin and γ-H2AX 0 and 24 h after Dox treatment in HUVECs pre-treated with control IgG or Ab417 (magnification, ×400). Scale bar = 10 μm. Error bars represent mean ± SD (IgG vs. Dox + IgG p = 0.0002; Dox + IgG vs. Dox + Ab417 p = 0.0482; Ab417 vs. Dox + Ab417 p = 0.0065). For quantification of γ-H2AX foci and γ-H2AX foci colocalized with L1CAM, the foci in each sample were counted at least 70 cells per field (magnification, ×100). The average number of foci/cell was determined from >6 fields (magnification, ×100). Data are representative of three independent experiments. (h: two-talied Student’s t-test, all other panels: one-way ANOVA for multiple comparisons).