Fig. 4

EBV-induced VM formation is HIF-1α-dependent. a Immunofluorescence of HIF-1α in TW03 and TW03-EBV cells with or without targeting of EBV by CRISPR/Cas9. Scale bars = 50 μm. b The mean integrated optical density (MOD) values of HIF-1α levels in the cytoplasm and nucleus of NPC cells with or without EBV. More than 100 cells were counted in randomly chosen fields. Mean ± SD, n = 3, two-tailed unpaired t-test. c Subcellular fractionation of EBV- cells, EBV+ cells and EBV+ cells subjected to EBV targeting by transfecting EBNA1 gRNAs. Each extract was analyzed by immunoblotting of HIF-1α, β-Tubulin, and PARP1. β-Tubulin represents cytoplasmic proteins, and PARP1 is used as a nuclear protein marker. d EBV− and EBV+ NPC cells were treated with 10 or 50 μM PX-478 for 24 h, followed by WB analysis of HIF-1α. e Immunofluorescence staining of HIF-1α in TW03-EBV cells that were treated with 10 μM PX-478 for 24 h or left untreated. Scale bars = 50 μm. f (left) Light microscopy of the tube formation of TW03-EBV cells that were pre-treated with 10 μM PX-478 for 24 h prior to plating on Matrigel for additional 12 h. Scale bars = 100 μm. (right) Quantification of the tube numbers formed by NPC-EBV cells after PX-478 treatment at different concentrations, as indicated. Mean ± SD, n = 5, two-tailed unpaired t-test. g EBV- and EBV+ TW03 cells were treated with 1 or 10 μM 2-MeOE2 for 12 h, followed by WB analysis of HIF-1α. h (left) In vitro tube formation of TW03-EBV cells that were treated with DMSO or 10 μM 2-MeOE2 for 12 h on Matrigel. Scale bars = 100 μm. (right) Quantification of tube numbers formed by NPC-EBV cells after 2-MeOE2 treatment. Mean ± SD, n = 5, two-tailed Mann–Whitney test. i Immunoblotting of NPC cells transfected with control or HIF-1α-specific siRNAs. j (left) Images and (right) quantification of tube formation of control and HIF-1α-depleted cells. Scale bars = 100 μm. Mean ± SD, n = 5, two-tailed Mann–Whitney test. ns: not significant, *p < 0.05, **p < 0.01 and *** p < 0.001