TNFα is internalized into RhoB-positive vesicles.

<p>(A) Cells were stained for RhoB before and after stimulation with TNFα for 4 hours (upper panels) and before and after proteasome inhibition with MG132 for 1 hour (lower panels); (B) Cells were stimulated with TNFα for 4 h and stained for RhoB (green) and EEA1 (red). Arrows point to vesicles positive for both proteins; (C) Cells were incubated at 4°C with biotin-TNFα and FITC-streptavidin (green) and transferred to 37°C for 30 min to allow internalization. Following fixation/permeabilization, cells were stained for RhoB (red). A magnification of the area within the box is shown on the right. Arrows point to vesicles where TNFα colocalises with RhoB. Bars: 10 µm.</p

RhoB regulates MAP kinase activation by TNFα.

<p>(A) Cells transfected with siRNA control or with a pool of RhoB siRNAs were stimulated or not with TNFα for 30 min. Lysates were prepared and incubated with an anti-phospho-MAP kinase antibody array. Pixel intensity of spots in the array was determined, corrected for background and represented as percentage of the positive controls included in the array; (B) Western blot analysis of phospho-ERK1/2, phospho-p38 and phospho-hsp27 in HUVEC transfected with siRNA control, with a pool of RhoB siRNAs (RhoB pool) or with single RhoB siRNAs (RhoB#1 and #2).</p

Pro-inflammatory mediators induce ‘de novo’ RhoB synthesis and RhoB activation in human umbilical vein endothelial cells.

<p>(A) Lysates of HUVEC incubated for 16 h with the indicated stimuli were analyzed for the expression of RhoB and RhoA by western blot. RhoGDI and tubulin were detected to control for equal loading; (B) Pull-down of GTP-Rho from HUVEC stimulated or not with TNFα for 4 h. Precipitates were analyzed for the presence of RhoB; (C) RhoB expression was induced by a 4 h TNFα stimulation, and subsequently proteasome inhibitor MG132 and/or the protein synthesis inhibitor cycloheximide was added to the cells for an additional 4 hours incubation; (D) RhoB detection in lysates of HUVEC stimulated with TNFα for 4 hours before the addition of cycloheximide (CHX) for 1, 2 or 4 hours. A digital scan of the film was made and the intensity of the RhoB bands was measured using ImageJ software. Data are shown as percentage of the RhoB present in the absence of cycloheximide. Unstimulated (solid blue line) and TNFα-stimulated cells (solid red line). Fitted regression lines obtained by linear regression analysis are shown as dotted lines; (E) Endothelial cells were incubated with TNFα in combination with the NFκB inhibitor sc-514, the JNK inhibitor SP600125, the p38 inhibitor SB230580 or the anti-oxidant N-acetyl cysteine (NAC). RhoB and VCAM-1 were detected in cell lysates. α-Tubulin was detected to control for equal loading.</p

RhoB silencing impairs TNFα-induced pro-inflammatory molecule expression.

<p>(A) Lysates of cells transfected with siRNAs as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075031#pone-0075031-g003" target="_blank">Figure 3</a> were stimulated with TNFα for 4 h and analyzed for total ICAM-1 and VCAM-1 expression by western blotting; (B) ELISA analysis of IL6 and IL8 present in conditioned media of cells transfected with a pool of RhoB siRNAs, with RhoA siRNA or a control siRNA were stimulated with TNFα for 4 or 16 h. Graph shows normalized values after dividing by the IL concentration in the medium of unstimulated siRNA control-transfected cells (1235±592 pg/mL IL8 and 187±7.5 pg/mL IL6) (mean ± SEM, n = 3; *<i>p</i><0,05).</p

IL-6 and IL-8 content of KLF2-transduced BOECs.

<p>(A) Immunofluorescence image showing co-localization of IL-6 (green) and VWF (red) in IL-1β-treated KLF2- and mock-transduced BOECs. Nuclei were visualized with DAPI (blue). Scale bars: 10 µm. (B) Western blot analysis for VWF, KLF2, IL-8 and IL-6 expression in lysates of mock- and KLF2-transduced BOECs; α-tubulin was shown as a loading control. (C) Immunofluorescence image showing co-localization of IL-8 (green) and VWF (red) in IL-1β-treated KLF2- and mock-transduced BOECs. Nuclei were visualized with DAPI (blue). Scale bars: 10 µm. (D) Release of VWF from PMA-stimulated KLF2 (black bars)- and mock (white bars)-transduced cells (IL-1β-treated), measured by determining the concentration of VWF in the conditioned medium by ELISA. **P<0.001; ***P<0.0001 by Students t-test (E-F) Release of IL-6 and IL-8 from PMA-stimulated KLF2 (black bars)- and mock (white bars)-transduced cells (IL-1β-treated), measured by determining the concentration of IL-6 and IL-8 in the conditioned medium by ELISA. The amount of IL-6 released without stimulation was slightly reduced in KLF2 expressing cells when compared to mock-transduced cells. NS: non-significant; *P<0.01; ***P<0.0001 by Students t-test.</p

OPG content of mock- or KLF2-transduced BOECs.

<p>(A) Immunofluorescence image showing co-localization of OPG (green) and VWF (red) in both mock- and KLF2-tranduced BOECs. Nuclei were stained using DAPI (blue). Scale bars: 10 µm.(B) Western blot analysis for VWF, KLF2, IL-8 and IL-6 expression in lysates of mock- and KLF2-transduced BOECs; α-tubulin was shown as a loading control.</p

Angiopoietin-2 content of WPBs in mock- and KLF2-transduced BOECs.

<p>(A) Immunofluorescence image showing the co-localization of Ang2 (green) and P-selectin (red) in WPBs of mock-transduced BOECs. Nuclei were visualized with DAPI (blue). Scale bars: 10 µm. (B) Western blot analysis of VWF, KLF2 and Ang2 expression in lysates of mock- and KLF2-transduced BOECs; α-tubulin is shown as a loading control. (C) Mock- and KLF2-transduced BOECs stained for VWF (red) and Ang2 (green). Nuclei were stained using DAPI (blue). Scale bars: 10 µm. (D-E) Release of Ang2 and VWF from PMA-stimulated KLF2 (black bars)- and mock (white bars)-transduced BOECs measured by determining the concentration of Ang2 in the medium by ELISA. **P<0.001; ***P<0.0001 by Students t-test. (F) Time course of regulated VWF and Ang2 secretion after PMA stimulation of mock- and KLF2-transduced BOECs.</p

Reduced average length of WPBs in KLF2-transduced BOECs.

<p>(A) Confocal microscopy analysis of WPBs in mock- and KLF2-transduced BOECs stained for VWF (red) and DAPI (blue). Scale bars: 10 µm. (B) Average amount of WPBs per cell in unstimulated and stimulated mock-transduced BOECs and KLF2-transduced BOECs. ***P<0.0001 using Student’s t- test (C) The average length of the WPBs in individual mock- or KLF2-transduced BOECs. WPBs from 20 randomly selected cells were analyzed. ***P<0.0005 by Student’s t-test (D) Release of VWF from forskolin/IBMX- and epinephrine/IBMX-stimulated KLF2 (black bars)- and mock (white bars)-transduced BOECs. ***P<0.0001 by Students t-test. Error bars represent SEM.</p

Expression of KLF2 in BOECs.

<p>(A) Immunofluorescent analysis of mock- and KLF2-transduced BOECs. Cells were immunostained for CD31 (red) and KLF2 (green); nuclei were stained using DAPI (blue). Scale bars: 20 µm; (B) Western blot analysis of KLF2 expression in lysates of KLF2-transduced and mock-transduced BOECs; α-tubulin is shown as a loading control.</p

HMHA1 colocalizes with RhoGTPases.

<p>(A-D) Colocalization of myc-tagged HMHA1 with endogenous Rac1 (A), Rac1 Q61L (B), Cdc42 G12V (C) and RhoA V14 (D) was studied by Confocal Laser Scanning Microscopy. Myc-tagged HMHA1 and HMHA-tagged Cdc42 and RhoA were detected by immunostaining in combination with detection of F-Actin. HMHA1 colocalized with endogenous Rac1 (A) and Rac1 Q61L (B) in membrane ruffles (arrows). A partial colocalization of HMHA1 with Cdc42 G12V (C) and RhoA V14 (D) was observed (arrows) although less clear than for Rac1. Higher magnification images of the boxed areas are included. Scale bars, 10 µm.</p