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Fig. 3 | Journal of Translational Medicine

Fig. 3

From: CHIP functions as an oncogene by promoting colorectal cancer metastasis via activation of MAPK and AKT signaling and suppression of E-cadherin

Fig. 3

CHIP promotes the migration and invasion abilities of DLD-1 cells in vitro and in vivo. a, b The cell migration curves between the siCHIP and sictrl cells (a) as well as hCHIP and ctrl cells (b) were detected using a real-time x-Celligence system. CIM-plate was seeded with 40,000 cells/well and the cell migration was continuous monitored for 24 h. Significant differences were indicated (Student’s t test,***P < 0.001). c Representative images and data of a Transwell migration and invasion assay for the establishing DLD-1 cell lines. The siCHIP and sictrl cells, as well as hCHIP and ctrl cells, were allowed to migrate through the transwell inserts [pre-coated with Matrigel (1:40) or not] for 24 h. Upper inserts were seeded with 40,000 cells/well in 150 μl RPMI-1640 media without serum. The lower chambers were filled with 600 μl RPMI-1640 media supplemented with 10% FBS. The number of migrated and invasive cells were fixed, stained, photographed, and compared with the control group. Each bar represents the mean ± SD. **P < 0.01, ***P < 0.001. All images were representative of at least three independent experiments with similar findings. d Would healing assay for the siCHIP and sictrl cells as well as hCHIP and ctrl cells. Confluent monolayers of the indicated cells were scraped with a pipette tip to generate wounds and then were cultured for 48 h. Representative images of wounds were recorded with the Light System Microscope IX71 at 0, 24, and 48 h. All images are representative of at least three independent experiments with similar findings. e Assessment of metastatic capacity of 5 × 106 DLD-1 cells stably expressing siCHIP, hCHIP and each control cells by inoculating nude mice via intraperitoneal injection. 6 weeks later, the mice were sacrificed, and metastatic nodules on the mesentery were collected. The numbers of the metastatic nodules were measured (***P < 0.001). f Whole cell extract of the metastatic nodules was prepared. The protein expression of CHIP was determined by western blotting. Actin was used as a loading control. g IHC analysis of CHIP expression in representative metastatic nodules (×200 magnification)

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