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. 2015 Jun 26;72(24):4849–4866. doi: 10.1007/s00018-015-1973-4

Fig. 5.

Fig. 5

Rab5a regulates the LPS-induced disruption of VE-cadherin and actin cytoskeleton remodeling in HPMECs. a The expression levels of VE-cadherin and F-actin in the cell membrane, cell cytosol fractions, and whole cells were determined by WB. Representative blots showing the expression of VE-cadherin in the membrane (upper panel), cytosol (2nd panel), and whole cells (3rd panel), F-actin (4th panel), and GAPDH (5th panel) are shown. HPMECs were transfected with or without Rab5a siRNA or the Rab5a WT plasmid and then treated with 1 μg/ml LPS for 6 h. b Quantitative data showing VE-cadherin protein levels in the cell membrane and cytosol, normalized to GAPDH expression. The data are presented as the mean ± SE n = 3. *p < 0.05 versus the membrane control group, ^ p < 0.05 versus the membrane LPS group; # p < 0.05 versus the cytosolic control group, $ p < 0.05 versus the cytosolic LPS group. c Localization of VE-cadherin in HPMECs following Rab5a siRNA or WT plasmid transfection. After pretransfection with NC siRNA, Rab5a siRNA, the NC plasmid, or the WT Rab5a plasmid, confluent HPMECs were stimulated with 1 μg/ml LPS for 6 h. VE-cadherin localization was detected by CLSM. Green, VE-cadherin; blue, DAPI. Arrows indicate intercellular gaps. Scale bar 25 µm. The photographs represent three independent experiments. d Quantification of the area of intercellular gaps as a percentage of the total area imaged. The data are presented as the mean ± SE based on three independent experiments. *p < 0.05 versus the NC siRNA group, ^ p < 0.05 versus the NC siRNA plus LPS treatment group. e Localization of the expression of F-actin in HPMECs following Rab5a siRNA or WT plasmid transfection. After pretransfection with NC siRNA, Rab5a siRNA, the NC plasmid, or the Rab5a WT plasmid, HPMECs were stimulated or not stimulated with 1 μg/ml LPS for 6 h. The actin cytoskeleton was stained with rhodamine-phalloidin and observed via CLSM. Red, F-actin; blue, DAPI. Arrows indicate stress fibers. Scale bar 25 µm. The photographs represent three independent experiments