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. 2024 Mar 5;9(7):e169830. doi: 10.1172/jci.insight.169830

Figure 7. CD93 regulates MMP9 levels in vitro as well as in primary and metastatic sites.

Figure 7

(A) Immunofluorescence images of MMP9 (green) in control mLECs (Mock and siCtrl) and mLECs silenced for CD93 (siCD93-4 and siCD93-5) with/without VEGF (10 ng/mL, 5 minutes). Actin and nuclei were visualized by phalloidin (red) and Hoechst (blue). Scale bars: 20 μm. (B). Quantification of MMP9 levels in mLECs. *P < 0.05, **P < 0.01 by 1-way ANOVA with Tukey′s multiple-comparison test (3 independent experiments). (C) Real-time qPCR showing Mmp9 mRNA levels in control mLECs (mock and siCtrl) and CD93-silenced mLECs (siCD93-4 and siCD93-5) with/without VEGF (3 independent experiments). **P < 0.01, ***P < 0.001 by 1-way ANOVA with Tukey′s multiple-comparison test. (D) Immunofluorescent staining of MMP9 (green) and CD31 (red) in HCmel12 primary tumor from wild-type and CD93–/– mice. Scale bars: 20 μm. (E) Quantification of the area covered by MMP9 normalized to the CD31+ area in wild-type (n = 5) and CD93–/– (n = 7) HCmel12 primary tumor tissue. (F) Immunofluorescent staining of MMP9 (green) and CD31 (red) in metastatic lungs from wild-type and CD93–/– mice. Metastatic lesion of mCherry-HCmel12 tumor cells (TC, gray) are defined by dotted line. Scale bars: 20 μm. (G) Quantification of the area covered by MMP9 around the lung metastatic lesion normalized to the CD31+ area in wild-type (n = 9) and CD93–/– (n = 9) lung metastatic lesions. All immunofluorescence quantifications were performed in a minimum of 4 fields of view/sample. AU, arbitrary units. *P < 0.05; **P < 0.01; ***P < 0.001 by 2-tailed t test (E and G). Values represent mean ± SEM.