Article Figures & Tables
Figures
- Fig. 1. The small GTPase Rac1 is activated by TNF-α and regulates the expression of VCAM-1 and ICAM-1.
HUVEC were treated with TNF-α as indicated and a Rac1.GTP (A), RhoG.GTP (B) and CDC42.GTP (C) pull-down assay was performed as described in Online Methods. Second and third panels show protein loading. Graphs on the right show quantification. All experiments described above were repeated at least four times. Data are mean±SD. *P<0.05. Rac1 (D), RhoG (E) and CDC42 (F) protein levels were reduced in HUVEC with lentivirally-delivered shRNA and TNF-α-induced VCAM-1 and ICAM-1 expression was examined. Actin is included for protein loading (lower panels). All experiments described above were repeated at least four times. Data are mean±SD. *P<0.01.
- Fig. 2. Trio expression in endothelial cells and in RA patients.
(A) Protein expression of GEFs Trio, Vav2, β-Pix, DOCK180, SGEF and Sos1 in TNF-α-treated HUVEC. (B) Trio and Vav2 mRNA levels examined by qRT-PCR in HUVEC after TNF-α. Data are means of four independent experiments±SEM. *P<0.05. (C) Trio protein expression in HUVEC was examined following TNF-α, IL-1β, LPS and IFNγ. Data are means of four independent experiments±SEM. *P<0.05; **P<0.05 (D) Trio protein expression was examined in A549 lung epithelial cells after TNF-α treatment. Graph shows quantification of Trio expression. (E) Immuno-histochemical staining of Trio in synovial biopsies of inflamed joints of patients with mild synovitis and rheumatoid arthritis (RA) patients. Arrowheads indicate Trio-positive endothelial cells. Asterisk indicates blood vessel lumen. Graph shows quantification of Trio-positive vessels in mild synovitis and RA patients. In total, 863 vessels from mild synovitis and 2695 vessels from RA patients were analyzed. Data are mean±SD. *P<0.01. (F) Immunofluorescent staining was carried out on samples from mild synovitis and RA patients. Overview shows merge of nuclei (blue), Trio (red) and ICAM-1 or VCAM-1 (green). Magnification shows localization of Trio and ICAM-1 or VCAM-1 at sites of vessels. Scale bars: 20 µm and (zoom) 10 µm.
- Fig. 3. Trio silencing reduces inflammatory cytokine-induced expression of ICAM-1 and VCAM-1 and reduces leukocyte adhesion and transendothelial migration.
(A) Five lentivirally-delivered shRNA constructs targeting Trio expression were examined for their potential to downregulate Trio protein expression. The GEF Vav2 and actin are shown as loading controls. (B) The effect of Trio silencing on the F-actin cytoskeleton in HUVEC that were stimulated with TNF-α. Immunofluorescent images show actin in green and nuclei in blue. Scale bars: 20 µm. (C) ICAM-1, VCAM-1 and E-selectin protein expression upon TNF-α time-course in shCTRL and shTrio-treated HUVEC. (D) ICAM-1, VCAM-1 and E-selectin mRNA levels by qRT-PCR in shCTRL and shTrio-HUVEC after TNF-α. Adhesion molecule mRNA levels in shTrio-treated HUVEC as a percentage of the adhesion molecule mRNA levels in controls. Data are means of three independent experiments ± SEM. (E) Upregulation of VCAM-1 (left graphs) and ICAM-1 (right graphs) by TNF-α, IL-1β, IFN-γ and LPS in shCTRL (open bars) and shTrio (closed bars)-treated HUVEC. Primary monocytes were perfused over HUVEC and adhesion (F) and TEM (G) was quantified as described in Materials and Methods. This experiment was carried out four times in duplicates. Data presented in all graphs are means±SEM. *P<0.01.
- Fig. 4. Trio and Rac1 activity are required for TNF-α-induced ICAM-1 and VCAM-1 expression.
(A) Trio and Vav2 activation following TNF-α treatment was examined by means of pull-down assays with GST-Rac1-G15A mutants as described in Methods</intraref> in SM. Total Trio and Vav2 expression in the cell lysates is shown as loading control. Rac1 activation (Rac1.GTP) after TNF-α stimulation is shown in the lower panels. This experiment was carried out four times. (B) Reduced Trio expression in endothelial cells (third panel) prevented Rac1 activation (first panel; Rac1.GTP) upon TNF-α treatment as well as VCAM-1 and ICAM-1 expression (lower panels). Graph at the right shows the quantification of Rac1.GTP levels in fold increase. The experiment was carried out at least three times. (C) Inhibition of TrioD1 activity using ITX3 showed impaired Rac1 activity (Rac1.GTP). Second panel shows equal Rac1 protein expression. Graph at the right shows the quantification of Rac1.GTP levels in fold increase. Experiment is performed three times. Data are mean±SEM. ITX3 (D) and NSC-23766 (E) prevented upregulation VCAM-1 and ICAM-1 after TNF-α, respectively. Experiments were carried out four times. Data presented in all graphs are means±SEM. *P<0.05.
- Fig. 5. TrioN partially rescues TNF-α-induced VCAM-1 expression in Trio-deficient cells.
(A) Trio expression was reduced in endothelial cells with shTrio5, resulting in impaired VCAM-1 and to a minor extent ICAM-1 expression after TNF-α treatment. GFP-TrioN was expressed in HUVEC by adenoviral delivery, as described in Online Methods. Transduction with adenovirally-delivered GFP was used as a control. Actin expression is shown for protein loading. (B) Graphs show quantification of VCAM-1 expression rescue by TrioN in Trio-deficient cells. The experiment was carried out four times. Data are mean±SEM. *P<0.05.
- Fig. 6. Trio depletion inhibits VCAM-1 promoter activity and prevents Ets-2 activity and translocation.
(A) VCAM-1 mRNA levels were measured as described in Materials and Methods. Left graph: untreated HUVEC; Right graph: TNF-α treated. Data are means of three independent experiments ± SEM. (B) VCAM-1 promoter activity in HUVEC (TNF-α 20 h) was measured as described in Materials and Methods. Data are means of three independent experiments ± SEM. *P<0.001. (C) HUVEC were treated as indicated and immunostained for Ets-2 in green and nuclei in blue. Profile on the right shows increase in fluorescence according to drawn line in image, and graph on the right shows quantification. Data are means of three independent experiments ± SEM. *P<0.01. (D) Trio was silenced in HUVEC, treated as described under C and GFP-TrioN (open bars) or GFP-TrioD1 (closed bars) was expressed. Ets-2 nuclear translocation was determined by fold increase in nuclear vs. cytosolic fluorescent signal. Data are means of three independent experiments ± SEM. *P<0.05. (E) Immunoprecipitation of Ets-2 showed increased threonine/serine (Thr/Ser) phosphorylation of Ets-2 upon TNF-α treatment, which was abrogated in Trio-deficient cells. Graph on the right shows quantification. Data are means of three independent experiments ± SEM. *P<0.01.
Article Navigation
Related articles
Cited by...
More in this TOC section
Similar articles
Other journals from The Company of Biologists
Biology Open and COVID-19
We are aware that the COVID-19 pandemic is having an unprecedented impact on researchers worldwide. The Editors of all The Company of Biologists’ journals have been considering ways in which we can alleviate concerns that members of our community may have around publishing activities during this time. Read about the actions we are taking at this time.
Please don’t hesitate to contact the Editorial Office if you have any questions or concerns.
Future Leader Review - Cardiac myosin super relaxation
A new Future Leader Review by Manuel Schmid and Christopher Toepfer discusses the rapidly-expanding field of myosin super relaxation in the context of cardiovascular disease. Read the full Review and their accompanying interview.
Find out more about our Future Leader Reviews – they are an exclusive opportunity for early-career researchers who want to establish themselves in their field.
An interview with Roberta Azzarelli
In an interview, first author Roberta Azzarelli discusses her 3D model of glioblastoma and shares her thoughts on how to improve the professional lives of early-career researchers: formal mentorship programmes, a clearly structured career path and taking part in initiatives such as the Node Network.
News from our sister journals
Development continues to run a successful new webinar series, Development presents…, while Journal of Cell Science has recently welcomed Esperanza Agullo-Pascual as FocalPlane’s new Community Manager. Journal of Experimental Biology’s new special issue highlights the role of comparative biology in tackling climate change and Liz Patton, the new Editor-in-Chief of Disease Models & Mechanisms, sets out her visions and priorities.