Fig. 7

ANXA5 induces the internalization of TLR4 via a PS-dependent entry. a PS externalization is present in LPS-activated HUVECs. Living HUVECs activated by LPS (1 μg/ml) were incubated with anti-PS antibodies. Then HUVECs were stained by AF488-conjugated secondary antibody. No LPS treatment as a negative control. Scale bars: 10 μm. b LPS-activated HUVECs were examined by TUNEL assay. Dnase I treatment as a positive control. c HUVECs were treated with LPS for different times. The exposed PS on activated HUVECs was detected by ANXA5-EGFP (50 μg/ml) and analyzed by FACS analysis. d Direct fluorescence observation of living HUVECs incubated with ANXA5/A5m-EGFP. After 2 h of ANXA5/A5m-EGFP incubation, HUVECs were observed by fluorescence microscope. e LPS-activated HUVECs were activated by LPS and then incubated with TLR4-FITC antibody. In the absence of ANXA5-TagRFP, the fluorescence signal of TLR4-FITC was spotty mainly on the plasma membrane. When the addition of ANXA5-TagRFP, TLR4-FITC was observed inside cells and co-localized with ANXA5-TagRFP. A5m-TagRFP as a negative control. f HUVECs were activated with LPS (1 μg/ml) for 0, 2, 4 h, and then treated with ANXA5 (250 nM) or A5m (250 nM) for 30 min. The surface TLR4 was stained by TLR4-FITC antibody for FACS. Data are presented as mean value of MFI representative for three independent experiments. g The activation of NF-κB in HUVECs treated with LPS and ANXA5 as indicated. P65 phosphorylation and IκB degradation were analyzed by western blot. h HUVECs were treated with different stimulation as indicated and were analyzed by western blot with antibodies against p65 and iκB. i HUVECs were transfected with NC siRNAs or TLR4 siRNAs. After stimulation with LPS and ANXA5, NF-κB activation was analyzed by western blot