Fig. 4
ERG regulates thrombomodulin expression and activity in vitro. a qPCR analysis of thrombomodulin (TM) mRNA expression in control (siCtrl) and ERG-deficient (siERG) HUVEC after 12, 24 and 48 h treatment (n = 3 independent experiments). Data were normalized to GAPDH. b Representative immunoblot and quantification of TM following siCtrl or siERG treatment on HUVEC for 12, 24 and 48 h (n = 4 independent experiments). Data were normalized to GAPDH. c Representative image and quantification of TM expression (green) in HUVEC transfected with siCtrl or siERG siRNA for 48 h by immunofluorescence; nuclei are identified by DAPI (blue) and cells are co- stained for ERG (magenta). Scale bar 40 µm. Quantification represents the mean pixel intensity for TM signal (arbitrary unit, A.U.) per cell. d, e Representative immunoblot and quantification of ERG and TM expression in control (siCtrl) and ERG-deficient (siERG) d HDMEC (microvascular EC) or e HDBEC (microvascular EC) after 48 h siRNA treatment (n = 3). f qPCR analysis of TM mRNA expression in HUVEC transfected with control pcDNA or ERG cDNA expression plasmid (ERG) (n = 3). Data were normalised to GAPDH. g siCtrl or siERG-treated HUVEC for 48 h were incubated with protein C (300 nM), CaCl2 (5 mM), thrombin (10 nM). After 10, 20, 30 and 60 min of incubation at 37 °C, anti-thrombin III (100 nM) and heparin (15 U per ml) were added to neutralise thrombin, and protein C activity was measured using chromogenic substrate S-2366 (0.5 mM) (n = 3). Data are expressed as relative activated protein C (APC) concentration normalised to siCtrl-treated condition following 60 min of incubation. All graphical data are mean ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test. Source data are provided as a Source Data file
