MDACl5exp cells did not show significant differences when compared to the control. In contrast, MDACL5rib2 cells demonstrated
a significant reduction in cell motility compared to the control (Figure 5a). The cells were additionally evaluated after treatment with HGF. This motogen increased cell motility in MDACl5exp and control cells when compared to untreated. In the case of MDACL5rib2, changes in motility were not found to be significant (Figure 5b). Figure 5 Effect of Claudin-5 on cell motility of MDA-MB-231 cells. (a) Cytodex-2 bead motility assay was used. The motility of MDA CL5rib2 was significantly reduced in comparison to the control MDA pEF6 (using one-tailed test, p = 0.027) (mean±SD, n = 3). (b) Effect on cell motility after treatment with HGF using a Cytodex-2 bead motility BIBW2992 in vivo assay. Transfected and control cells showed an increase in motility, however only MDA Cl5exp results were significant (p ≤ 0.001 versus respective untreated cells) (mean±SD, n = 3). (c) Effect of Claudin-5 on cell migration was assessed by a migration/wound healing assay. MDACL5expcells showed
an increase in migration when compared to the control at 60 minutes after wounding (*p ≤ 0.005) (mean ± SD, Palbociclib clinical trial n = 3). The migration of MDACl5rib2 was reduced in comparison to the control at 60 minutes (**p ≤ 0.005) (mean ± SD, n = 3). (d) Significant differences using ECIS were revealed after wounding. MDACL5exp showed significant increased migration (p ≤ 0.001) whereas MDACl5rib2 showed a decreased migration rate (p ≤ 0.001) (n = 3). The effect of Claudin-5 on cell migration was assessed using an in vitro cellular migration/wound healing assay. MDACl5exp showed a
significant increase in cellular migration compared to the control 60 minutes after. A significant decreased cell migration was seen in MDACL5rib2 after 60 minutes when compared to control (Figure 5c). In this assay, we are investigating the direct movement of cells as they migrate from a 4-Aminobutyrate aminotransferase cell layer into open space. The cytodex-2 bead assay in comparison, measures the motility of single cells. It is not surprising that the over-expression or knock-down of Claudin-5 appears to be more significant in the wounding assay; it appears that Claudin-5 might be involved in the signalling pathway for changes in contact inhibition and changes in the cytoskeleton, rather than in simple motility (as assessed using the bead assay). Using ECIS (Electrical Cell Impedance Sensing) and in recovering from electrical wounding (5 V AC for 30 seconds), it was shown that the MDACl5exp cells were significantly more motile compared to the control cells as the resistance in the electrode increased as the cells begin to spread over the electrode, whereas the opposite trend was seen in MDACL5rib2, where a significant reduction in migration was seen (Figure 5d).