Skip to main content
. 2015 May 13;290(27):16653–16664. doi: 10.1074/jbc.M115.636944

FIGURE 2.

FIGURE 2.

CoCl2, a mimic of hypoxia, induces endothelial to mesenchymal transition. A, representative bright field images showing the morphology of untreated and CoCl2-treated cells (200 and 400 nm). CoCl2-treated HCAECs showed a fibroblast-like phenotype. Scale bars, 25 μm. B, Western blot analysis showing expression of HIF1α, CD31, VE-cadherin, α-SMA, and SNAIL in HCAECs treated with CoCl2 (200 and 400 nm). All blots were reprobed with an anti-actin antibody as a loading control. C, representative immunofluorescence images showing CD31 (red) and α-SMA (green) staining in untreated (left panel) and CoCl2-treated cells (right panel); nuclei were counterstained with DAPI (blue). Acquisition of a spindle-shaped morphology upon CoCl2 exposure correlated with increased α-SMA expression and loss of CD31. Scale bars, 20 μm. D, untreated control cells (left panel), cells treated with 200 nm CoCl2 (middle panel), or cells treated with 400 nm CoCl2 (right panel) were sorted by FACS according to CD31 expression. CD31 protein was labeled with phycoerythrin (PE) (red) (x axis). In control cells, CD31+ cells were most prominent (gate Q4). This population was decreased under CoCl2 treatment in favor of CD31 cells (gate Q3). E, quantification of CD31+ and CD31 cells exposed to CoCl2 treatment. F, cells from gate Q3 and Q4 of 400 nm CoCl2-treated cells were sorted and compared by quantitative real time PCR analysis. Expression of mRNAs encoding the endothelial markers (CD31, VE-cadherin (VE-Cad), and VWF) and mesenchymal markers (FSP1, α-SMA, DDR2, and collagen 1A1) in the CD31 (Q3) cell population is shown compared with their expression in the CD31+ (Q4) cell population. G–J, qRT-PCR data showing the mRNA expression of EndMT transcription factors (SNAIL, SLUG, and TWIST) and COL1A1 in untreated and CoCl2-treated cells. EndMT transcription factors and COL1A1 were significantly induced in endothelial cells upon CoCl2 treatment. Results were normalized to reference gene GAPDH (expression is presented as mean value; error bars represent S.D.; n = 3; **, p < 0.01; ***, p < 0.001). K, representative immunofluorescence images showing CD31/Hif1α (red) and SNAIL (green) in normoxic (upper panel) and hypoxic cells (lower panel); nuclei were counterstained with DAPI (blue). Acquisition of HIF1α upon CoCl2 treatment correlated with SNAIL acquisition. Scale bars, 20 μm.

HHS Vulnerability Disclosure