FIGURE 1.

S100B stimulates BV-2 microglia migration in a RAGE-dependent manner. A, migration assays were performed using Boyden chambers. The cells (1 × 10⁵) were seeded onto the top of Transwell® migration chambers and allowed to migrate for 6 h toward S100B or medium alone negative control placed in the lower chamber. B, conditions were as in A except that BV-2 or rat primary microglia were pretreated with nonimmune IgG or a RAGE neutralizing antibody for 60 min and then transferred to the upper chamber. C, conditions were as in A except that S100B (0–1.0 μm) was added to the cells placed on the upper well of Boyden chambers, where indicated. D, conditions were as in A except that WT BV-2 microglia (BV-2/WT), mock-transfected BV-2 microglia (BV-2/mock), BV-2 microglia stably expressing a RAGE mutant lacking the cytoplasmic and transducing RAGE domain (BV-2/RAGEΔcyto), or BV-2 microglia stably overexpressing full-length RAGE (see “Experimental Procedures”) were used. E, BV-2 microglia were cultivated in DMEM for 6 h in the presence of increasing S100B concentrations. The culture media were collected, trichloroacetic acid-precipitated as described under “Experimental Procedures,” and subjected to Western blotting using an anti-HMGB1 antibody. Residual cells were lysed, and cell lysates were probed with anti-HMGB1 antibody. Also shown is a Western blot of tubulin. F, BV-2 microglia were pretreated with BoxA and allowed to migrate for 6 h toward medium alone placed in the lower chamber. The results are expressed as the means ± S.D. (n = 3) (A–D, F, and G). G, mouse WT and Rage^−/− microglia were subjected to migration assay as described in A in the presence of increasing S100B concentrations. *, significantly different from control (first columns in A, C, F, and G) or from internal control (first columns from left in each group in B and D). #, significantly different from the corresponding column in the BV-2/WT + IgG group or the primary microglia group in B and in the BV-2/mock group in D.