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. 2021 Jun 25;12(7):649. doi: 10.1038/s41419-021-03920-4

Fig. 4. Effects of TNF-α on TIE1 and TIE2 receptors.

Fig. 4

A TNF-α decreases the mRNA and protein expression of TIE1 and had no effect on TIE2 expression. HMVECs were treated various times with 100 ng/ml of TNF-α. mRNA and protein levels were quantified by RT-qPCR and western blot, respectively; graphs represent protein quantifications. RT-qPCR histograms show the mean of three independent biological experiments, and western blots are representative of three independent biological experiments. B, C The overexpression of TIE1 induces a delay of TNF-α-induced EndMT (B) and increases TIE1/TIE2 interactions (C). Control HMVECs and TIE1-encoding lentivirus infected clone (ST1) were treated various times with 100 ng/ml of TNF-α. Scale bars, 100 µm (inset) or 2000 µm. The graph represents the variation rate of proteins in presence of TNF-α. Lysates from control HMVECs and overexpressing TIE1 ST1 cells were subjected to immunoprecipitation with an anti-TIE2 or anti-MYC antibody, and TIE1 was detected by western blot. The arrow shows an aspecific band. TBP for RT-qPCR and β-tubulin for western blot analysis were used as controls. Data are representative of three independent experiments. Significant differences are indicated by solid lines (***P < 0.005 by t test). HMVEC human microvascular endothelial cell, EndMT endothelial–mesenchymal transition, TNF-α tumour necrosis factor-α, ST1 cells overexpressing TIE1, N-Cad N-cadherin, Ctrl control, mRNA messenger RNA, TBP TATA-box binding protein.