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

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

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

DataSheet_1_Oxygen level is a critical regulator of human B cell differentiation and IgG class switch recombination.pdf

The generation of high-affinity antibodies requires an efficient germinal center (GC) response. As differentiating B cells cycle between GC dark and light zones they encounter different oxygen pressures (pO2). However, it is essentially unknown if and how variations in pO2 affect B cell differentiation, in particular for humans. Using optimized in vitro cultures together with in-depth assessment of B cell phenotype and signaling pathways, we show that oxygen is a critical regulator of human naive B cell differentiation and class switch recombination. Normoxia promotes differentiation into functional antibody secreting cells, while a population of CD27++ B cells was uniquely generated under hypoxia. Moreover, time-dependent transitions between hypoxic and normoxic pO2 during culture - reminiscent of in vivo GC cyclic re-entry - steer different human B cell differentiation trajectories and IgG class switch recombination. Taken together, we identified multiple mechanisms trough which oxygen pressure governs human B cell differentiation.</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

Visualization and flow cytometry analysis of endogenous HMHA1 using ImageStream.

<p>(A) Jurkat T-cells were fixed and immunostained for endogenous HMHA1 and Rac1 and stained for F-actin and DNA. Left panel shows three examples of the distribution of HMHA1, Rac1 and F-actin revealing colocalization of HMHA1 and Rac1 in F-actin rich areas. The nucleus (DNA) and cell morphology (phase image) are included to show the integrity of the cell. Right panel shows intensity distribution of Rac1 (Y-axis) and HMHA1 (X-axis) signals, underscoring the fact that most cells are double positive. (B) Jurkat cells were stimulated for the indicated time-points with 100 ng/ml CXCL12 and analyzed as in A. Two examples of each condition are shown in the left panels. Changes in F-actin distribution in response to CXCL12 can be observed, in particular after 1 and 5 minutes. Right panels show the extent of colocalization (AU, arbitrary units) quantified by the image stream software. Ave, average colocalization, n, number of cells.</p

The HMHA1 GAP domain negatively affects cell spreading.

<p>Cell spreading was measured by Electrical Cell-substrate Impedance Sensing (ECIS) following seeding of 100.000 cells on fibronectin-coated electrodes. Left panel: A significant decrease in electrical resistance, as a measure of cell spreading, was observed in HeLa cells expressing HMHA1 C1-GAPtail (black), C1-GAP (light green), and GAPtail (grey) compared to control cells (red). Ectopic expression of HMHA1 full-length (blue), N-term (dark green), and GAP (magenta) did not affect cell spreading. Right panel: Relative cell spreading at 60 minutes post-seeding. Data are mean values of three independent experiments. Error bars indicate SEM. ns, not significant, ** p<0.01, *** p<0.001.</p

HMHA1 is a RhoGAP <i>in vitro</i>.

<p>(A) Sequence alignment of HMHA1 with the typical RhoGAP, p50RhoGAP, and the structurally-related BAR-GAPs, GRAF1 and OPHN1. Green indicates two matching amino acids. Pink indicates three matching amino acids. Purple indicates four matching amino acids. The arginine finger region is indicated with a black bar. (B) 3D model of the protein-protein complex between RhoA and the HMHA1 RhoGAP domain highlighting the catalytic residues (in sticks, colour coding as indicated; P-loop-Switch I-Switch II of RhoA in light green). The homology model for the GAP domain of human HMHA1 is based on the structure of the human p50RhoGAP domain (PDB ID: 1tx4), using Phyre. The position of the HMHA1 GAP domain in the complex with human RhoA (from RhoA⋅GDP⋅AlFx⋅p50RhoGAP; PDB ID: 1tx4) was obtained through its overlay on the p50RhoGAP domain. The RhoGAP domain of GRAF1 from <i>Gallus gallus</i> (PDB ID: 1f7c) was superimposed onto the model of the HMHA1 GAP domain. (C) HMHA1 C1-GAPtail has <i>in vitro</i> GAP activity towards Rac1, Cdc42, and RhoA but not towards Ras (purple bars). p50RhoGAP was used as a positive control (red bars). GTPases or HMHA1 only were used as a control and as a measure for intrinsic nucleotide hydrolysis (yellow bars). Data are mean values of two independent experiments. Error bars indicate SD. (D) HMHA1 GAP activity is inhibited by the N-terminal BAR domain as full-length HMHA1 has no GAP activity while C1-GAPtail, lacking the N-terminal region, shows GAP activity (purple bars). GTPases or HMHA1 only were used as a control and as a measure for intrinsic hydrolysis (yellow bars). Data are mean values of two independent experiments. Error bars indicate SD.</p

Localization and effects on F-actin of HMHA1 and selected mutants.

<p>Intracellular localization of myc-tagged HMHA1 (and mutant constructs) was studied by Confocal Laser Scanning Microscopy following expression in HeLa cells. Myc-tagged HMHA1 was detected by immunostaining for the myc epitope in combination with detection of F-Actin with phalloidin. Full-length HMHA1 (FL) as well as HMHA1 N-term are partially localized at membrane ruffles as well as in the cytoplasm. For HMHA1 N-term localization at vesiculo-tubular structures is occasionally observed (arrows). Cells expressing FL or the N-term constructs are morphologically similar to control cells and no effects are seen on F-Actin (upper two rows). HMHA1 constructs lacking the C-terminal tail (GAP and C1-GAP) are partly mislocalized into protein aggregates. In cells expressing HMHA1 C1-GAP, C1-GAPtail, and GAPtail (marked with asteriks), F-Actin distribution is altered and cell morphology is dramatically changed. Higher magnification images of the boxed area are included. Scale bars, 10 µm.</p