Effects of rhChM-I on the invasion of Rcho-1 trophoblast cells. (A) Effects of rhChM-I on the production and activation of MMP-9 in cultures of Rcho-1 trophoblast cells. Cells were grown to confluence, and the culture medium was then replaced with differentiation medium (NCTC-135 medium containing 10% horse serum). At the indicated time points (at day 2, 4, 6, 9, and 13), the culture medium was changed to medium containing 1% horse serum and conditioned for 24 h. The gelatinolytic activity in the conditioned medium was analyzed by gelatin zymography (upper panel). rhChM-I or GM6001 (25 μM) was added to cultures of Rcho-1 trophoblast cells in differentiation medium on day 9, and the cells were incubated another for 24 h in medium containing 1% horse serum. The gelatinolytic activity in the conditioned medium was then analyzed by gelatin zymography (lower panel). The gel images are representative of three independent experiments. (B) Matrigel invasion assay of Rcho-1 cells. Subconfluent Rcho-1 trophoblast cells were harvested and resuspended in NCTC-135 medium containing 0.5% horse serum, and seeded onto cell culture inserts (5 × 104 cells/200 μl) coated with a layer of Matrigel. The cells were allowed to invade for 16 h in the presence of 0.1% BSA/PBS (control), various concentrations of rhChM-I, or GM6001 (25 μM) in NCTC-135 medium containing 0.5% horse serum. The number of cells that had invaded the undersurface of the insert was counted under × 200 magnification. The values shown are percentages of the number of invading cells compared with the control cells (361 ± 20.7 cells/field) and are the means ± SD of a triplicate assay. The data are representative of three independent experiments with similar results. The statistical significances were determined by the Student's t-tests as compared to the control (0.1%BSA/PBS) (*P < 0.05, one-tailed). (C) Representative microphotographs of Rcho-1 trophoblast cells that had invaded the bottom surface of the insert in the presence of 0.1% BSA/PBS (upper panel, control) or 1.5 μg/ml rhChM-I (lower panel). Bars, 100 μm.
Miura et al. BMC Cell Biology 2011 12:34 doi:10.1186/1471-2121-12-34