Fig 2: COUP-TF2 directly targets CCND1 and NRP1 to mediate cell proliferation and migration.(A) ChIP followed by qPCR was performed to amplify the target region in the Ccnd1 and Nrp1 promoters. (B) qPCR analysis of COUP-TF2 in WT and COUP-TF2 KO iMVECs. (C) Immunoblotting of CCND1 and NRP1 in both WT and KO iMVECs, numbers under Western blot bands represent relative quantifications over actin. n = 2. (D) CCK-8 assay showing cell proliferation of iMVECs treated ±VEGFA165 (20 ng/ml) for 72 hours. n = 4. (E) Cell migration was assessed using a wound scratch assay. Images were obtained at (0 hours) and (24 hours). Representative photos illustrate scratch closing, quantified in the graph (right). Scale bar, 100 µm. (F) Immunoblotting analysis of indicated proteins in WT, COUP-TF2-KO, and COUP-TF2-OE iMVECs treated ±VEGFA165 (20 ng/ml) for 30 min, demonstrating VEGFR phosphorylation dependent on COUP-TF2 levels. Numbers under Western blot bands represent relative quantifications over actin. n = 2. (G) Graphical abstract. Each dot represents one independent experiment. Data are presented as means ± SD. *P < 0.05 and **P < 0.01, calculated by unpaired two-tailed t test.

Fig 3: COUP-TF2 is inhibited by TNF-a– and IL-1ß–mediated NF-?B activation in vitro.(A) qPCR analysis of COUP-TF2 expression in iMVECs treated with either TNF-a or IL-1ß. (B) Western blot analysis of COUP-TF2 expression in iMVECs treated with either TNF-a or IL-1ß. Numbers under Western blot bands represent relative quantifications over actin. n = 3. (C) ChIP-qPCR was performed to amplify the target region in the COUP-TF2/Nr2f2 promoter. iMVECs were pretreated for 2 hours with NF-?B inhibitors BAY-117082 (5 µM) and JSH-23 (5 µM), followed by addition of either TNF-a or IL-1ß for 24 hours. qPCR analysis of COUP-TF2 in both TNF-a (D) and IL-1ß (E) treatment group. (F) qPCR analysis of NF-?B target genes E-selectin, ICAM-1, and VCAM-1 in isolated lung ECs (CD45-CD31+) sorted on days 0 (uninjured), 10, and 27. Each dot represents one independent experiment or one mouse. Data in (A), (D), and (E) are presented as means ± SD. Data in (C) and (F) are presented as means ± SEM. Data in (A), (C), (D), and (E) were calculated using unpaired two-tailed t test; data in (F) were calculated using two-way ANOVA, followed by Dunnett’s multiple comparison test. *P < 0.05 and **P < 0.01. TSS, transcriptional start site.

Fig 4: NR2F2 regulates WJ-MSC proliferation and cell cycle. (A) Growth curves of WJ-MSCs under control RNAi and NR2F2 RNAi conditions. Each time point in each row is n = 4. (B) Immunocytochemical staining for DAPI (blue) and Ki67 (green). (C) Flow cytometry to examine cell cycle changes. (D) Cell apoptosis was detected by flow cytometry. (E) qPCR results, relative expression levels of CCND1, CDK4, and CDK6 genes in control (control RNAi) and NR2F2 knockdown (NR2F2 RNAi) groups. (F) Western Blot analysis of cyclin CCND1, CDK4 expression. * p = 0.05, ** p = 0.01, *** p = 0.001.

Fig 5: Global gene expression changes upon NR2F2 knockdown in WJ-MSCs. (A) WJ-MSCs exhibited two distinct gene expression patterns after NR2F2 was knocked down. (B) Each point in the volcano plot represents a gene. Red dots indicate significantly up-regulated genes, blue dots indicate significantly down-regulated genes, and grey dots indicate non-differentially expressed genes. (C) Heatmap showing the top 50 genes with the most significant up- and down-regulation, with red for up-regulation and green for down-regulation. (D) The darker the red of the Hub gene, the higher the score.