Fig. 3: An anti-L1CAM antibody prevents p53 knockout- and radiation-induced DNA damage in heart ECs. | Nature Communications

Fig. 3: An anti-L1CAM antibody prevents p53 knockout- and radiation-induced DNA damage in heart ECs.

From: An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage

Fig. 3

a RT-qPCR analysis of L1CAM in p53-knockdown HUVECs 48 h post-irradiation (10 Gy). Error bars represent mean ± SD from three independent experiments (p < 0.0001). b Immunoblotting (upper panel) and quantification (lower panel) of full-length L1CAM and L1-CT 48 h post-irradiation (10 Gy) in p53-knockdown HUVECs pre-treated with control IgG or Ab417 (20 µg/mL). Error bars represent mean ± SD from four independent experiments (full-length L1CAM: No IR vs. si-Control IR + IgG p = 0.0468; si-Control IR + IgG vs. si-Control IR + Ab417 p = 0.0468; si-Control IR + IgG vs. si-p53 IR + IgG p = 0.0462; si-p53 IR + IgG vs. si-p53 IR + Ab417 p = 0.0218, L1-CT: No IR vs. si-Control IR + IgG p = 0.0124; si-Control IR + IgG vs. si-Control IR + Ab417 p = 0.0484; si-Control IR + IgG vs. si-p53 IR + IgG p = 0.0206; si-p53 IR + IgG vs. si-p53 IR + Ab417 p = 0.0004). c Immunofluorescence staining (upper panel) and quantification (lower panel) for γ-H2AX and L1-CT. The results of quantification of γ-H2AX foci, nuclear L1CAM, and colocalization of γ-H2AX foci with L1CAM are shown (magnification, ×400). Scale bar = 5 μm. Error bars represent mean ± SEM (No. γ-H2AX foci: si-Control vs. si-p53+IgG p = 0.0107; si-p53+IgG vs. si-p53 + Ab417 p = 0.0242, No. L1-CT foci: si-Control vs. si-p53+IgG p = 0.0425; si-p53+IgG vs. si-p53 + Ab417 p = 0.0119, No. colocalized foci: si-Control vs. si-p53 + IgG p = 0.0131; si-p53 + IgG vs. si-p53 + Ab417 p = 0.0097). For quantification of foci colocalized with L1CAM, the colocalized foci in each sample were counted in at least 70 cells per field (magnification, ×100, n = 5). d, e Wild-type or EC-p53KO mice were injected intravenously with control IgG or Ab417 (10 mg/kg) and subjected to 17.5 Gy thoracic irradiation (n = 5 animals per group). d Immunofluorescence staining (upper panels) for γ-H2AX, L1CAM, and CD31 in heart tissues 3 weeks post-irradiation and quantification (lower panels) of γ-H2AX+ cells and nuclear L1CAM+ cells among CD31+ cells (magnification, ×400). Scale bar = 5 μm. Error bars represent mean ± SEM (γ-H2AX+ in CD31+ nuclei: WT+IR vs. p53KO+IR p = 0.0005; p53KO + IgG vs. p53KO + Ab417 p < 0.0001, L1CAM+ in CD31+ nuclei: WT+IR vs. p53KO+IR p = 0.0001; p53KO+IgG vs. p53KO + Ab417 p = 0.0002). e Haematoxylin and eosin staining, Masson’s trichrome staining, and immunohistochemical detection of CD31 in heart tissues 3 weeks post-irradiation (upper panels) and quantification of ventricular inflammation, perivascular fibrosis area, and microvessel density per field (magnification, ×200; lower panels). Scale bar = 100 μm. Error bars represent mean ± SEM (ventricular inflammation: No IR vs. WT IR + IgG p = 0.0003; WT IR + IgG vs. WT IR + Ab417 p = 0.0125; WT IR + IgG vs. p53KO IR + IgG p = 0.0104, perivascular fibrosis area: No IR vs. WT IR + IgG p = 0.0021; WT IR + IgG vs. WT IR + Ab417 p = 0.0328; WT IR + IgG vs. p53KO IR + IgG p = 0.0203; WT IR + IgG vs. WT IR + Ab417 p = 0.0165, MVD: No IR vs. WT IR + IgG p < 0.0001; WT IR + IgG vs. WT IR + Ab417 p = 0.0055; WT IR + IgG vs. WT IR + Ab417 p = 0.0068). f Immunofluorescence staining (left panels) of α-SMA and CD31 in heart tissues 3 weeks post-irradiation (n = 5 animals per group) and quantification (right panels) of the α-SMA+CD31+ area in the CD31+ area (magnification, ×400). Scale bar = 20 μm. Error bars represent mean ± SEM (No IR vs. WT IR + IgG p < 0.0001; WT IR + IgG vs. WT IR + Ab417 p = 0.0251; WT IR + IgG vs. p53KO IR + IgG p = 0.011; p53KO IR + IgG vs. p53KO IR + Ab417 p = 0.0004). g Immunofluorescence staining (left panel) of WGA, cTnT, and CD31 (n = 5 animals per group) and quantification (right panel) of the cTnT area per field (magnification, ×400). Scale bar = 20 μm. Error bars represent mean ± SEM (No IR vs. WT IR + IgG p = 0.0014; WT IR + IgG vs. WT IR + Ab417 p = 0.0489; WT IR + IgG vs. p53KO IR + IgG p = 0.0253; p53KO IR + IgG vs. p53KO IR + Ab417 p = 0.001). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant (a Two-way ANOVA for multiple comparisons; all other panels: one-way ANOVA for multiple comparisons).

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