Dose-dependent inhibition of chemotaxis and cell migration by LAI-1.

D. discoideum amoebae harboring pSW102 (GFP) were treated for 1 h with different concentrations of (A) racemic LAI-1, (B) 10 μM (R)-LAI-1, (S)-LAI-1, (R)-amino-LAI-1 or (S)-amino-LAI-1, or (C) different concentrations of CAI-1, and cell migration towards folate (1 mM) was monitored in under-agarose assays for 4 h. Graphs depict per cent GFP fluorescence intensity versus migration distance. (D) D. discoideum amoebae harboring pSW102 (GFP) were treated with LAI-1 (10 μM, 1 h). Single cell migration towards folate (1 mM) was monitored in under-agarose assays for 15 min. Motility parameters (forward migration index, FMI; and velocity) were analyzed using the ImageJ manual tracker and Ibidi chemotaxis software. (E) Murine RAW 264.7 macrophages were treated for 1 h with different concentrations of racemic LAI-1, cell migration towards CCL5 (100 ng/ml) was monitored in under-agarose assays for 4 h, and the cells were stained with Cell Tracker Green BODIPY. Macrophages treated for 1 h with 10 μM LAI-1 were immuno-labeled for (F) α-tubulin (green) or (G) actin (red) and, as a control, the production of cellular α-tubulin or actin was quantified by Western blot. Microtubule fibers per cell were counted along cross-sections (S3 Fig), and the actin architecture was analyzed by quantifying the number of cells displaying cortical actin. The graphs show means and standard deviations of 3 independent experiments (n > 25 (α-tubulin) or > 40 (actin) single cells; Student´s t-test, *p < 0.05, **p < 0.01). Bars (F, G), 5 μm.