Table 1.
Effect of VEGF and VEGF-Trap on MT dynamic instability parameters in HUVECs.
Figure 1.
(A) Localization of EB1 in HUVECs by indirect immunofluorescence after 1 h of VEGF treatment at various concentrations. Bar, 10 µm. (B) Dose response effect of VEGF on EB1 comet length. * p≤0.001 (vs control, Student t test). (C) Incubation of HUVECs with VEGF trap (1 h) inhibited VEGF-increase of EB1 comet length. * p≤0.001. (D) Western blotting detection of EB1 and tubulin in HUVECs after 1 h of treatment with VEGF at various concentrations.
Figure 2.
VEGF induced EB1 C-terminal −EEY modification and loss of CAP-Gly protein interaction.
(A) Effect of VEGF on EB1 focalization on 2D gel electrophoresis. Cells were incubated for 1 h with VEGF (10 ng/ml) or vehicle alone. Indicated experimental isoelectric points were calculated according to reference protein (stathmin). (B) Detection of −EEY sequence with YL½ antibody by 2D gel electrophoresis of HUVECs under control and VEGF (10 ng/ml) treatment. YL½ antibody allowed the detection of the −EEY sequence both in tubulin and EB1. Revelation of EB1 using anti-EB1 antibody on the same membrane after stripping is presented for each condition ( left of panels). (C) Localization of p150Glued in HUVECs treated or not with VEGF (10 ng/ml) by indirect immunofluorescence. Bar = 10 µm. (D) Western blotting detection of p150Glued and tubulin in HUVECs after 1 h of treatment with VEGF at various concentrations or VEGF Trap.
Figure 3.
Detection of detyrosinated EB1 in HUVECs and U87 cells.
(A) HUVECs incubation with 3-L-nitrotyrosine allowed detection, in total cell lysates, of a nitrotyrosinated form of tubulin but also of EB1. Representative of 3 independent experiments (B) Purified tyrosinated and detyrosinated EB1 (EB1-EEY, EB1ΔY) proteins were loaded as, respectively, negative and positive probes in regard to detyrosinated EB1 detection in HUVECS and U87 total cell lysate by using a specific guinea pig anti-detyrosinated EB1 antibody [22] (C) Transwell migration assay was performed in U87 cells depleted of EB1 (clone sh11) and expressing GFP-EB1 full length or GFP-EB1ΔY mutant. U87 sh0 clone was also used as a negative control. Data show the average number of cells that migrated. At least three independent experiments were performed for each condition. Bar ± S.E.M. (*) indicates significant differences from controls: *, p<0.05; **, p<0.005. (D) Western blot was performed with down-regulating EB1 U87 sh11 clone transfected or not with GFP-EB1 full length, GFP-EB1ΔY expression plasmids or control GFP-empty vector. U87 sh0 clone was also used as a negative control. Tyrosinated-EB1 was detected by using a specific anti-EB1 tyrosinated antibody [22].
Figure 4.
Anti-migratory vinflunine decreased EB1 comet length.
(A) Number of migrating HUVECs in transwells following VFL treatment. *p≤0.001 (vs control, Student t test). (B) Concentration-dependent inhibition of cell survival by VFL after 72 h of treatment (C) Dose-response effect of VFL on EB1 comet length. * p≤0.05 (vs control, Student t test). (D) Correlation between EB1 comet length and the mean number of migrating cells/well.
Figure 5.
Anti-migratory vinflunine favored the tyrosinated form of EB1.
(A) Effect of VFL on EB1 focalization on 2D gel electrophoresis. Cells were incubated for 5 h with VFL (10, 100 nM) or vehicle alone. Indicated experimental isoelectric points were calculated according to reference protein (stathmin) (B) Detection of EB1-EEY sequence with YL½ antibody increased in a concentration-dependent manner under VFL concentrations. Relative ratios are presented under the blots. (C) Correlation between detection of EB1-EEY sequence and reduction of EB1 comet length by VFL.
Figure 6.
Anti-migratory vinflunine altered EB1 detyrosination/retyrosination cycle in both endothelial and glioblastoma cells.
Western blotting detection of detyrosinated or tyrosinated EB1 in HUVECs (A) and U87 cells (B) under VFL treatment. Relative ratios detyrosinated EB1 (EB1ΔY)/total EB1 or tyrosinated EB1 (EB1-EEY)/total EB1, from at least five independent experiments, are presented under the blot. (C) Transwell migration assay with control sh0 U87cells treated or not with VFL (10 nM). Data show the average number of cells that migrated through the filter. At least three independent experiments were performed for each condition. Bar ± SEM (**) indicates significant differences from control (p<0.05).