Figure 3.

Effect of PTx on PDT-induced monocytes adhesion, migration and protein tyrosine phosphorylation. (A, B) Static adhesion assay of monocytes on ICAM-1. Cells pretreated with 500 ng/mL of PTx for 2 h at 37 °C were stimulated for 2 min at 37 °C with PBS (NT), fMLP (100 nM), PDT (10 µg/mL) (A) or for 10 min with PMA (100 ng/mL) (B). Bars represent the means ± SD of 3 independent experiments performed in triplicate. Statistical analysis was performed by paired 2-tail Student t test, *** p < 0.001, * p < 0.05. (C) Transwell migration assay of monocytes in response to the indicated treatments. Monocytes pretreated with PTx were stimulated for 90 min at 37 °C with PBS (NT), LPC (10 µM), fMLP (10 nM) or PDT (0.5 µg/mL). Bars represent the means ± SD of 3 independent experiments performed in triplicate. Statistical analysis was performed by paired 2-tail Student t test, *** p < 0.001. (D) Monocytes pretreated or not with PTx were stimulated or not for 5 min at 37 °C with PDT (5 µg/mL). Western blot analysis of cell lysates shows that PDT-triggered protein tyrosine phosphorylation was inhibited by PTx, as shown by densitometry analysis and plotting of the pTyr/tERK1/2. In the left panel blots from one representative experiment of three with similar results are shown. In the right panels, values reported for protein Tyr phosphorylation are the mean ± SD of three independent experiments. Statistical analysis was performed by paired 2-tail Student t test and the Bonferroni’s post-test was used to compare data, *** p < 0.001. NT = not treated.