Fig 2: Evaluation of Dll4 as a predictor of disease activity in patients with non-renal SLE and LN. (A) ROC curves of the sensitivity and specificity of Dll4 in patients with non-renal SLE and LN. (B) ROC curve of the sensitivity and specificity of Dll4 in patients with active LN. (C, D) Dll4 immunofluorescence staining of renal sections in the control group (healthy normal kidneys of renal cancer patients) and LN patients. Scale Bar = 20 μm, n = 3, ***P <0.001.

Fig 3: Dll4 promotes the proliferation and migration of MC via Dll4 in vitro. (A) Co-culture illustration of ECs and MCs according to different treatment, the control group was treated with LPS to ECs alone, siRNA-Dll4 group was siRNA-Dll4 in ECs then treated with LPS, Tarextumab group was ECs treated with LPS and MCs treated with Tarextumab. (B) qRT-PCR results of the Dll4 mRNA expression after Dll4 siRNA transfection in ECs (n = 3, *P < 0.05, **P < 0.01). (C) Transwell assay showing the migration of MC in different groups, Scale bar = 2 mm (upper), Scale bar = 300 μm (lower). (D) MC migration statistics (n = 3, **P < 0.01, ***P < 0.001). (E) EdU assays were performed to characterize the proliferation of MC when knocking down Dll4 in ECs or adding Tarextumab. Scale bar = 150 μm. (F) MC proliferation statistics n = 3 for each group, **P < 0.01, ***P < 0.001.

Fig 4: Interaction network between ECs and other cell types in the kidneys of LN mice. (A) Heatmap of cell expression correlation between EC subtypes and other cell types. The color denotes the correlation coefficients between the ligand-receptor pair and the cell types. (B) Ligand-receptor relationship between MCs and EC subclusters, when MCs were the receptor cells. (C) Bubble plot showing the expression of Dll4 in EC subclusters. (D) Co-immunolocalization of Dll4 and Pi16 on EC-1, and co-immunolocalization of Notch3 and Fn1 on MCs. Scale bar = 50 μm, n = 3.

Fig 5: EC regulate MC via the Dll4/Notch3 axis. (A) ELISA assays were performed to detect the expression of Dll4 in ECs after LPS stimulation. (B) CCK8 assay showing the effects of Dll4 on MC viability. (C) qRT-PCR assay showing Notch3 relative genes were upregulated in MC after Dll4 stimulation. (D) CCK8 assay showing the effects of Tarextumab on MC viability after Dll4 interference. (E) Western blotting analysis of the expression of the Notch3-ICD and its downstream-related proteins HEY1 and HEY2 following interference with Dll4 alone or in combination with Tarextumab. (F) Quantification of Western blotting. Data were representative of three independent experiments (n = 3 per group). (G) Representative images of wound healing assays showing the migration of MC cells treated with Dll4 or Dll4 in combination with Tarextumab. Scale bar = 200 μm. (H) Quantitative comparison of MC cell migration rates among Control, Dll4, and Dll4+Tarextumab treatment groups (n = 3). (I) Transwell analysis of the effect of Dll4 and Tarextumab on MC cell migration. Scale bar = 2 mm for overview. Scale bar = 300 μm for magnified view. (J) Quantitative analysis of the number of migrated cells. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01. (K) EdU assays were performed to detect the proliferation of MC treated with Dll4 and Tarextumab. Scale bar = 200 μm. (L) Quantitative analysis of MC cell proliferation by EdU assay. Data are presented as mean ± SD (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.