Figure 3
EVs derived from GD2+ S1 cells markedly enhanced the cell growth and invasion of GD2- V4 cells. (A, B) Proliferation was analyzed using GD2- V4 cells and GD2+ S1 cells by MTT assays. Cells (3 × 103) were seeded in each well of 96-well plates. Then, EVs (0.5, 2.0 and 4.0 μg) derived from S1 cells were added to V4 cells (A), and EVs (0.5, 2.0 and 4.0 μg) derived from V4 cells were added to S1 cells (B). PBS alone was added to control cells. MTT solution (5 mg/ml in PBS) was added and incubated at 37 °C for 4 h on each day as indicated. After vigorous pipetting, the absorbance was measured at 590 nm, and the relative absorbance was plotted. The analysis was performed in triplicate, and the mean ± SD is presented. (A, B) The data at day 1, 3, 5 and 7 were analyzed by two-way ANOVA with a Tukey post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. (C–F) Invasion was examined with cell culture inserts using GD2+ S1 and GD2- V4 cells. The upper chamber of the inserts was coated with 200 μg/ml Matrigel. After 24 h of incubation, noninvaded cells were removed, invaded cells in the lower chamber were stained with Giemsa, and the number of cells was counted under a microscope. (C, E) Microscopic images of invaded cells from the upper chambers where GD2- V4 and GD2+ S1 cells were plated, followed by the addition of S1-derived EVs and V4-derived EVs, respectively. (D, F) The analysis was performed in triplicate, and the mean ± SD is presented. The data were analyzed with an unpaired Student’s two-tailed t test. ***P < 0.001. Scale bar: 20 μm.
