Fig 2: Suppression of cell invasion and migration induced by metastasis associated in colon cancer 1 (MACC1) short hairpin (sh)RNA. (A) Wound healing assay was conducted after silencing of MACC1 expression. (A, upper panel; scale bar=200 µm) Migration rate was significantly inhibited in the shRNA(MACC1) cells, as determined by a wound healing assay, and (A, lower panel) the migration rate was quantified. **P<0.01, vs. shRNA(-) group. (B, upper panel; scale bar=100 µm) Transwell assay following silencing of MACC1 expression (B, lower panel) and the comparison of invasion capacity in the U251, shRNA(-) and shRNA(MACC1) cells. **P<0.01, vs. shRNA(-) group. (C) Expression levels of invasion and migration-associated proteins. (C, upper panel) Protein expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 in the U251, shRNA(-), and shRNA(MACC1) cells were tested and compared by western blot analysis; (C, lower panel) the quantitative analysis of gray intensity was calculated. **P<0.01, vs. shRNA (-) group. (D) The activities of MMP-2 and MMP-9 were decreased by MACC1 shRNA. (D, upper panel) The activities of MMP-2 and MMP-9 were tested by gelatin zymography assay; and (D, lower panel) the relative activities of MMP-2 and MMP-9 were calculated. **P<0.01, vs. shRNA(-) group. Data are presented as the mean ± standard deviation. U251, U251 cells without RNA interference; shRNA(-), U251 cells transfected with negative control MACC1 shRNA; shRNA(MACC1), U251 cells transfected with silenced MACC1 shRNA.

Fig 3: Silencing of metastasis associated in colon cancer 1 (MACC1) expression inhibited cell proliferation, and induced cell cycle arrest in U251 human glioma cells. (A) Colony formation rate was decreased by MACC1 silencing in small hairpin (sh)RNA(MACC1) U251 cells. (A, upper panel) The number of colonies in the shRNA(MACC1) cells was much lower, as compared with the number of colonies in the U251 and shRNA(-) cells. (A, lower panel) Colony forming efficiency was calculated. **P<0.01, vs. shRNA(-) group. (B) Suppression of cell proliferation by MACC1 shRNA in the U251 cells was measured using an MTT assay. The proliferation of U251 cells was significantly inhibited in a time-dependent manner following MACC1 silencing. **P<0.01, vs. shRNA(-) group. (C) Cell cycle arrest in the U251 cells was induced by MACC1 shRNA. Changes in the U251 cell cycle induced by MACC1 shRNA were examined using fluorescence-activated cell sorting. (C, upper panel) There was an increase in shRNA(MACC1) cells at G0/G1 phase; (C, lower panel) The ratio of U251, shRNA(-) and shRNA(MACC1) cells in G0/G1 phase was calculated. *P<0.05, vs. shRNA(-) group. (D) Expression levels of cell cycle arrest-associated proteins cyclin D1, cyclin E and cyclin B were examined and compared by western blot analysis, and (E) the quantitative analysis of gray intensity was calculated. *P<0.05, **P<0.01, vs. shRNA(-) group. Data are presented as the mean ± standard deviation. U251, U251 cells without RNA interference; shRNA(-), U251 cells transfected with negative control MACC1 shRNA; shRNA(MACC1), U251 cells transfected with silenced MACC1 shRNA.

Fig 4: Connection diagram of MACC1, c-Met, and cyclin D1.

Fig 5: OS and PFS of ESCC patients after chemotherapy were analyzed by the Kaplan–Meier method. Notes: (a) OS according to postoperative chemoradiotherapy (P = 0.001); (b) PFS according to postoperative chemoradiotherapy (P = 0.01); (c) OS according to MACC1 expression (P = 0.03); (d) OS according to c-Met expression (P = 0.003); (e) PFS according to c-Met expression (P = 0.03). Abbreviations: OS: overall survival; PFS: progression-free survival; ESCC: esophageal squamous cell carcinoma.