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. 2020 Jun 23;11:593. doi: 10.3389/fphys.2020.00593

FIGURE 6.

FIGURE 6

Coculture of hUCMSCs and VEGF-induced hUCMSCs (V-hUCMSCs) activated the expression of the HIF-1 signaling pathway in V-hUCMSCs. The differentially expressed genes of V-hUCMSCs (V) and cocultured V-hUCMSCs (VC) from the RNA-seq results are shown using a heatmap (A). A volcano plot summarizing the RNA-seq data indicating the differentially regulated transcripts between V and VC (B) and a gene set enrichment analysis (GSEA) plot of the Hif-1 signaling pathway (C). qRT-PCR analysis showed that coculture of hUCMSCs and V-hUCMSCs increased the mRNA (VEGFA, TIMP1, EDN1) expression in V-hUCMSCs. The relative gene expression was normalized to the expression of β-actin, and the control was set to 1.0. Values are expressed as the mean ± SD (***P < 0.001) (D). Western blot was used to detect the expression of VEGFA and Hif-1α in all V and VC cells. β-actin was used as an internal control (E). Western blot demonstrated that coculture significantly upregulated Hif-1α expression in the nucleus. Histone3 and β-actin were used as internal controls (F). Immunofluorescence staining revealed that the expression of Hif-1α was increased in the nucleus in VC (G).