Volume 122, Issue 2, Pages 331-339 (February 2002) Tumor necrosis factor α stimulates invasion of Src-activated intestinal cells  Naoki Kawai, Shingo Tsuji, Masahiko Tsujii, Toshifumi Ito, Masakazu Yasumaru, Yoshimi Kakiuchi, Arata Kimura, Masato Komori, Yutaka Sasaki, Norio Hayashi, Sunao Kawano, Raymond Dubois, Masatsugu Hori  Gastroenterology  Volume 122, Issue 2, Pages 331-339 (February 2002) DOI: 10.1053/gast.2002.31023 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Phenotypes of RIE transfectants. (A) Morphologic features of RIE-m, RIE-ras, and RIE-src cells. (B) The protein levels of Ras and Src. The cells were cultured on the plastic plates in DMEM/1% FCS for 24 hours. (C) Response of RIE-src cells to TNF-α in the presence or absence of PDTC and herbimycin A. RIE-src cells cultured on plastic plates were treated as described in Materials and Methods. The bar represents 100 μm (magnification, 100×). Gastroenterology 2002 122, 331-339DOI: (10.1053/gast.2002.31023) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Colonies of RIE-ras and RIE-src cells in soft agar. Vehicle or TNF-α (40 ng/mL) treatment was started 2 days after cells were embedded in soft agar. The pictures were taken on day 7. (A) RIE-ras control, (B) RIE-ras treated with TNF-α, (C) RIE-src control, (D) RIE-src treated with TNF-α. Gastroenterology 2002 122, 331-339DOI: (10.1053/gast.2002.31023) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Comparison in sizes of colonies of RIE-ras and RIE-src cells in soft agar. The cells were cultured in soft agar in the presence of vehicle or TNF-α for 7 days, and then the longest diameters were measured. PDTC, herbimycin A, a Src-specific inhibitor, or AFC, a ras inhibitor, was added with TNF-α. These figures are the representative of 5 independent experiments. *P < 0.01 compared with nontreated value, **P < 0.01 compared with TNF-α–treated value. Gastroenterology 2002 122, 331-339DOI: (10.1053/gast.2002.31023) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Activation of Src TK. (A) Status of tyrosine phosphorylation of Src. Cells were pretreated for 1 hour with PDTC (10 μmol/L) or herbimycin A (0.5 μg/mL), then treated for 3 hours with vehicle or TNF-α, and harvested. Src was precipitated from the cell lysates (400 μg) by use of anti-Src antibody, and tyrosine phosphorylation and Src protein were detected by immunoblot analysis. (B) Activity of Src protein tyrosine kinase. c-Src immunoprecipitates were collected the same as above. Protein tyrosine kinase activity was measured by enzyme-linked immunosorbent assay. These figures are the representative of 5 independent experiments. *P < 0.01 compared with nontreated value, **P < 0.01 compared with TNF-α–treated value. Gastroenterology 2002 122, 331-339DOI: (10.1053/gast.2002.31023) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 FACS showing peroxide levels in RIE-ras and RIE-src cells treated with TNF-α. After staining with DCFH-DA, the cells were treated with TNF-α for 1 hour. Pretreatment with PDTC preceded TNF-α treatment by 1 hour. Intracellular peroxide level was estimated by FACS. Panels A, B, and C represent RIE-m, RIE-ras, and RIE-src cells, respectively. Lines indicate as below: (a) the control, (b) TNF-α–treated, and (c) PDTC-pretreated groups. In RIE-m cells, line (c) was overlapped with (a). Gastroenterology 2002 122, 331-339DOI: (10.1053/gast.2002.31023) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Regulation of E-cadherin expression and tyrosine phosphorylation of β-catenin and E-cadherin. Cells were incubated in DMEM/1% FCS in the presence or absence of vehicle or TNF-α for 8 hours. Pretreatment with PDTC or herbimycin A preceded TNF-α treatment by 1 hour. (A) Comparison of E-cadherin expression among RIE transfectants. Whole cell lysates (20 μg) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and E-cadherin protein was detected by anti–E-cadherin antibody. The same blot was stained with Coomasie brilliant blue. (B) mRNA level of E-cadherin. Total RNA was prepared from the cells treated with vehicle or TNF-α for 5 hours, and E-cadherin mRNA level was detected by RT-PCR analysis. (C) Status of tyrosine phosphorylation of β-catenin and E-cadherin. Whole cell lysates (400 μg) associated with E-cadherin were collected by immunoprecipitation using anti–E-cadherin antibody and subjected to immunoblot analysis by use of anti-phosphotyrosine or β-catenin antibody. Gastroenterology 2002 122, 331-339DOI: (10.1053/gast.2002.31023) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Comparison of motility of RIE transfectants. Cells were suspended in DMEM/0.1% bovine serum albumin, plated on Boyden chamber inserts (pore size 8 μm) with vehicle or TNF-α, and incubated for 8 hours. In the bottom chambers, laminin (50 μg/mL) was added as a chemoattractant. PDTC, herbimycin A, AFC, or TGF-α was added to the inserts 1 hour before TNF-α treatment. These figures are the representative of 5 independent experiments. *P < 0.01 compared with nontreated value, **P < 0.01 compared with TNF-α–treated value. Gastroenterology 2002 122, 331-339DOI: (10.1053/gast.2002.31023) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Comparison of invasion among RIE transformants. Cells were incubated on the Matrigel matrix loaded on the invasion chamber inserts for 10 hours, and the medium in the insert and the bottom chamber was replaced with DMEM/1% FCS and vehicle or TNF-α. The assays were started with addition of hepatocyte growth factor (20 ng/mL) in the bottom chamber and incubated for 24 hours. The numbers of cells that invaded through the filters of inserts were counted. PDTC, herbimycin A, AFC, or TGF-α was added 1 hour before TNF-α treatment. These figures are the representative of 4 independent experiments. *P < 0.01 compared with nontreated value, **P < 0.01 compared with TNF-α–treated value. Gastroenterology 2002 122, 331-339DOI: (10.1053/gast.2002.31023) Copyright © 2002 American Gastroenterological Association Terms and Conditions