TialFig. 3. (a) To demonstrate that rac-4 also inhibits VCAM-1 expression at
TialFig. 3. (a) To demonstrate that rac-4 also inhibits VCAM-1 expression at low-non-toxic concentrations, HUVEC were stimulated with TNF- for 24 h inside the PDE3 Purity & Documentation presence or absence of diverse concentrations of rac-4. Note that at these concentrations inhibition of VCAM-1 occurs. VCAM-1 expression was assessed by Western blotting, -actin was utilized as loading handle. (b) HUVEC have been grown in 96-well plates until confluency and subsequently incubated with serial dilutions (000 mM) of rac-1 (graph towards the left) or rac-8 (graph to the ideal). Cell viability was assessed at unique time points (24, 48 and 72 h) by MTT as described. All experimental situations have been tested in triplicates in no less than five independent experiments. nnP o0.01 with respect to untreated cells. (c) Cells have been stimulated with TNF- for the indicated time periods inside the presence or absence of 50 mM of rac-1, L1 (panels to the left), rac-8 or L2 (panels for the suitable). Compound L3 (Fig. 1) as an additional doable hydrolysis/α1β1 web disintegration solution of rac-8 was tested in several experiments and gave similar final results as L2 (data not shown). Cells that were not stimulated with TNF- served as handle. VCAM-1 expression was assessed by Western blotting; -actin was applied as loading control. (d) Cells have been stimulated with TNF- for five days in the presence or absence of 25 or 12.5 mM of rac-1 or rac-8. Cells that weren’t stimulated with TNF- served as handle. VCAM-1 expression was assessed by Western blotting; -actin was utilised as loading manage (panel to the left). HUVEC had been grown in 96-well plates till confluency and subsequently incubated with 12.5 or 25 mM of rac-1 or rac-8. Cell viability was assessed by MTT assay (panel towards the right) and was expressed as viable cells relative towards the untreated cells. All experimental situations were tested in triplicates in no less than five independent experiments. (e, f) HUVEC have been stimulated for 24 h with TNF- (10 ng/ml). Hereafter, 50 mM of rac-1 (e) or rac-8 (f) was added without changing the medium and the cells had been cultured for further 24 h. VCAM-1 expression was assessed at 24 h of TNF- stimulation to assure that it was present prior to addition of rac-1 or rac-8 and soon after 48 h to test if addition of rac-1 or rac-8 was nevertheless capable to impact VCAM-1 expression. Cells that did not get rac-1/rac-8 served as control. Cells that weren’t stimulated with TNF were integrated to demonstrate VCAM-1 induction (panels to the left). In separate experiments cells were stimulated for 24 h with TNF- (ten ng/ml) inside the presence or absence of 50 mM of rac-1 or rac-8. Soon after 24 h in separate wells the medium was exchanged for medium that only contained TNF- (10 ng/ml) (removal) or medium that contained both TNF- and rac-1 or rac-8 (presence) and cells were allowed to grow for more 24 h. VCAM-1 expression was assessed at 24 h to demonstrate that rac-1 inhibits VCAM-1 expression and immediately after 48 h to demonstrate that VCAM-1 expression reappeared just after removal of rac-1 and rac-8 too. Cell cultures grown for 48 h in the continuous presence of TNF- (c) and cells that weren’t stimulated with TNF- were also included (panels to the right). For (c) to (f) information of a representative experiment are shown. At least four independent experiments happen to be performed with essentially the same outcomes.E. Stamellou et al. / Redox Biology 2 (2014) 739Fig. 3. (continued)cellular uptake of rac-1 and rac-4 is most likely not underlying the variations in cytotoxicity as these differe.
