Figure 4. Effect of CnMVs on lipid raft activation and fusion of HBMECs.

HBMECs were treated with PBS or purified CnMVs (0.5, 1, or 4 µg) for 2 h. The cells were lysed in a buffer containing 1% Triton X-100 on ice, and the cell lysates were fractionated in Optiprep™ gradients. Nine fractions were collected. The lipid raft fractions were located in Fraction 2, indicated by a red arrow. An equal volume of each sample was analyzed by dot blots with antibody against CD44 (A) or protein blots with antibodies against caveolin-1, β-actin, and vimentin (B). For cell fusion studies, D54GM cells were treated with the Celltracker red dye CMTPX (1^st panel) or the green dye CMFDA (2^nd panel) and then incubated with 10 µg purified CnMVs. Syncytial cells were detected after 6 hours of co-culture and displayed a bright yellow signal (yellow arrow) (C). Images shown in (D) are a confocal microscopic section with split images: CMFDA dye (green), CMTPX dye (red), DPAI (blue), and the overlaid image (D). Images shown in (E) are a confocal image with a top and two side views.