Figure 1.
TNFα induces migration and invasion of HCT116 cells.
(A) HCT116 cells were allowed to migrate transwell chambers for 24 h in the presence or absence of TNFα (20 ng/ml). After 24 h, the migrated cells were fixed, stained, and photographed. Magnification, 100×. (B) The number of migrated cells. Data represent the average of three independent experiments. (C) After treatment with or without TNFα (20 ng/ml) for 48 h, HCT116 cells that had spread through the matrixgel and into the under-side of the filter were fixed, stained, and photographed. Magnification, 200×. (D) The number of invasive cells. Data represent the average of three independent experiments. *p<0.05 compared with control.
Figure 2.
TNFα treatment triggers EMT in HCT116 cells.
(A) HCT116 cells were treated with or without TNFα (20 ng/ml) for 4 days. Cell morphological changes associated with EMT are shown in the phase contrast image. Expression of E-cadherin, ZO-1, fibronectin, N-cadherin were analyzed by immunofluorescence staining. Nuclei were visualized with DAPI staining. Scale bars: 20 µm. (B) HCT116 cells were treated with or without TNFα (20 ng/ml) for 4 days, and the expression of E-cadherin, ZO-1, fibronectin, N-cadherin, Snail, ZEB1, Twist, Slug were analyzed by western blotting. β-actin servers as the loading control. (C) HCT116 cells were treated with or without TNFα (20 ng/ml) for 4 days. The mRNA levels of E-cadherin, N-cadherin, Snail, ZEB1, Twist, Slug were analyzed by qRT-PCR. *p<0.05 compared with control.
Figure 3.
Snail is crucial for TNFα-induced EMT.
(A) pcDNA-Snail (Snail) or control vector pcDNA-3.1 (Vector) were expressed in HCT116 cells for 48 h. Cell morphological changes associated with EMT are shown in the phase contrast image. Expression of E-cadherin and N-cadherin were analyzed by immunofluorescence staining. Nuclei were visualized with DAPI staining. Scale bars: 20 µm. (B) The expression of E-cadherin, N-cadherin and Snail from HCT116 cells transfected with pcDNA-Snail or control vector were examined by western blotting. β-actin servers as the loading control. (C) HCT116 cells transfected with Snail specific si-RNA (si-Snail) or negative control si-RNA (si-NC) were stimulated with or without TNFα (20 ng/ml) for 48 h, and the morphologic changes were observed with a phase-contrast microscopy. (D) HCT116 cells transfected with si-Snail or si-NC were stimulated with or without TNFα (20 ng/ml) for 4 days, and the expression of E-cadherin and Snail were detected by western blotting. β-actin servers as the loading control.
Figure 4.
TNFα regulates Snail stabilization and localization.
(A–B) HCT116 and Caco-2 cells were treated with TNFα (20 ng/ml) for the times indicated, and the protein (A) and mRNA (B) levels of Snail were examined by western blotting and qRT-PCR respectively; (C) HCT116 cells were treated with or without TNFα (20 ng/ml) for 6 h. After fixation, the cellular location of Snail (green) was examined by immunofluorescence staining and nuclei were stained with DAPI (blue). Scale bars: 20 µm.
Figure 5.
TNFα stabilizes Snail through AKT/GSK3β
pathway. (A) HCT116 cells were pretreated with SB-203580 (20 µM), PD98059 (20 µM), BAY11-7082(10 µM), LY294002 (20 µM) for 1 h respectively followed by stimulation with TNFα (20 ng/ml) for 6 h. The expression of Snail was examined by western blotting. Caco-2 cells were pretreated with SB-203580 (20 µM), BAY11-7082(10 µM), LY294002 (20 µM) for 1 h respectively followed by stimulation with TNFα (20 ng/ml) for 6 h. The expression of Snail was examined by western blotting. (B) HCT116 cells were pretreated with or without LY294002 (20 µM) for 1 h, followed by stimulation with or without TNFα (20 ng/ml) for 6 h. The expression of Snail and the activation of AKT and GSK-3β were examined by western blotting. (C) HCT116 and Caco-2 cells were treated with TNFα (20 ng/ml) for the times indicated. The expression of pGSK-3β and GSK-3β were examined by western blotting. (D) Control and GSK-3β si-RNA were expressed in HCT116 cells for 42 h, followed treated with or without TNFα (20 ng/ml) for additional 6 h. The expression of Snail and GSK-3β were examined by western blotting. (E) HCT116 cells were treated with TNFα (20 ng/ml) or LiCl (40 mM) for 6 h. The expression of Snail, pGSK-3β, GSK-3β, and β-catenin were analyzed by western blotting. (F) After treated HCT116 cells with or without TNFα (20 ng/ml) for 6 h, Snail and β-catenin located at membrane and nuclear were isolated respectively and then analyzed by western blotting.
Figure 6.
TNFα inhibits the association of Snail and GSK3β.
(A) HCT116 cells were treated with TNFα (20 ng/ml) or MG132 (10 µM) for 6 h. After Snail was immunoprecipitated from equal amount of lysates (two lower panels), the ubiquitination of Snail was examined by western blotting. (B) HCT116 cells were treated with TNFα (20 ng/ml) or MG132 (10 µM) for 6 h. Snail or GSK-3β were immunoprecipitated respectively from equal amount of lysates and the associated GSK-3β or Snail were detected by western blotting.