Fig. 5
From: Vasculogenic mimicry formation in EBV-associated epithelial malignancies
EBV promotes VM formation via the PI3K/AKT/mTOR/HIF-1α axis. a Immunofluorescence of phospho-AKT (S473) in TW03 cells and TW03-EBV cells with or without targeting EBV by CRISPR/Cas9. Scale bars = 50 μm. b Immunoblots comparing AKT phosphorylation in EBV- and EBV+ NPC cell lines. c CNE2-EBV cells were transfected with empty vector or a dominant-negative mutant of EBNA1 (dnEBNA1), followed by WB analysis using antibodies as indicated. d Immunoblots of AKT phosphorylation and HIF-1α expression following LY294002 (50 μM) or Wortmannin (1 μM) treatment for 6 h. e Immunoblots of HIF-1α distribution in cytoplasmic and nuclear fractions of EBV- and EBV+ NPC cells following LY294002 (50 μM), Wortmannin (1 μM) or DMSO treatment for 6 h. f Immunofluorescence of phospho-AKT (S473) and HIF-1α in TW03-EBV cells after 6 h of treatment with DMSO, LY294002 (50 μM) or Wortmannin (1 μM). Scale bars = 50 μm. g Microscopy of tube formation on Matrigel in TW03-EBV cells after 12 h of treatment with LY294002 (50 μM) or Wortmannin (1 μM). Scale bars = 100 μm. h Quantification of tube numbers formed by NPC-EBV cells following LY294002 or Wortmannin treatment. Mean ± SD, n = 5, two-tailed unpaired t-test. i Effects of PI3K/mTOR/S6K kinase inhibitors on EBV-induced HIF-1α expression. CNE2-EBV cells were treated with DMSO, 1 μM Wortmannin (Wort), 0.5 μM rapamycin (Rapa) or 10 μM PF-4708671 (PF) for 6 h under normoxic conditions prior to immunoblotting with antibodies, as indicated. j Effect of rapamycin (0.5 μM) on VM channel formation of CNE2-EBV cells. Mean ± SD, n = 5, two-tailed unpaired t-test. ns: not significant, *p < 0.05, ***p < 0.001
