Figure 4

Cytokines induce filopodia production in BV-2 cells. (A) Time course for cytokine-induced filopodia formation in BV-2 cells. Representative bright field photomicrographs were taken with an inverted Nikon microscope (20×). Red arrows show processes with a fan-like ending. (B) Counting cells containing filopodia at 4 h after exposure to individual cytokines, LPS or combination as indicated. Results are expressed as % of filopodia cells versus total cell numbers (see Methods). Results are mean ± SEM from 4 independent experiments. Results are analyzed by one-way ANOVA followed by Dunnett's multiple comparison test, **p < 0.01 vs. control. (C) Staining F-actin in BV-2 cells after treatment with IFNγ and/or MEK1/2 inhibitor U0126. BV-2 cells were cultured in coverslip and serum starved for 4 h. Cells were pretreated with U0126 for 30 min prior to exposure to IFNγ for 4 h. Cells were then fixed with 4% paraformaldehyde and permeabilized by 0.1% Triton X-100 in PBS as described in text. After blocking non-specific binding with 5% normal goat serum (NGS), cells were incubated in rhodamine-phalloidin (1:100) and then mounted onto microscope slides and examined using the Leica DMI4000 automatic epifluorescence microscope with 40× objective lens. Space bar: 20 μm. White arrows denote filopodia. (D) Bar graph representing filopodia-containing cells after incubation with/without IFNγ, U0126, and U0126 + IFNγ. Two-way ANOVA revealed a significant interaction (p = 0.009) between U0126 and IFNγ, and a significant effect of U0126 (p < 0.0001), and IFNγ (p < 0.0001).