1. Volume 128, Issue 4, Pages 1002-1011 (April 2005)
Clostridium difficile toxin B activates the EGF receptor and the ERK/MAP kinase pathway in human colonocytes 
Xi Na, Dezheng Zhao, Hon Wai Koon, Ho Kim, Johanna Husmark, Mary P. Moyer, Charalabos Pothoulakis, J. Thomas Lamont 
Gastroenterology 
Volume 128, Issue 4, Pages (April 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
TxB-induced phosphorylation of EGFR and ERK. (A) NCM460 cells were incubated with TxB (20 nmol/L) for various times. Cell lysates were resolved on SDS-PAGE gels and immunoblotted against phosphorylated EGFR and ERK1/2. Antibodies against total EGFR or ERK1/2 were used to show equal protein loading. (B) NCM460 cells were seeded on collagen-coated permeable supports. TxB (20 nmol/L) was added to either apical or basolateral compartments for 1 hour and the activation of EGFR and ERK1/2 was examined (as described in A). (C) NCM460 cells were exposed to different concentrations of TxB (1.0–40.0 nmol/L) for 1 hour. Both phosphorylated and total EGFR and ERK1/2 were detected. (D) HT29 cells were incubated with TxB (20 nmol/L). Activations of EGFR and ERK1/2 were measured (as described in A). (E) NCM460 cells were exposed to TxA (20 nmol/L) for different time periods. Activations of EGFR and ERK1/2 levels were determined as described in A. The Western blots shown here are representative of 3 individual experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
Activation of ERK1/2 is dependent on EGFR phosphorylation. (A) NCM460 cells were pretreated with AG1478 at indicated concentrations for 30 minutes and then exposed to TxB (20 nmol/L) or TxA (20 nmol/L) for 60 minutes. Equal amounts of cell lysates were loaded on the SDS-PAGE gels and immunoblotted using antibodies against phosphorylated EGFR and ERK1/2. Total EGFR and ERK1/2 were detected as loading controls. (B) NCM460 cells were preincubated with control IgG (20 μg/mL) or neutralizing antibodies against EGFR (20 μg/mL) for 30 minutes and then exposed to TxB (20 nmol/L) for 1 hour. EGFR and ERK1/2 activation were detected as described earlier. Results are expressed as mean ± SEM of 3 experiments per group. *P < .05, cells pretreated with neutralizing antibodies against EGFR vs cells pretreated with control IgG in TxB-exposed cells. (C) NCM460 cells were preincubated with PTX (100 ng/mL) overnight and then exposed to TxB (20 nmol/L) for 60 minutes or lysophosphatidic acid (lpa, 25 μmol/L) for 10 minutes. ERK1/2 phosphorylation was detected as described earlier. (D) NCM460 cells were preincubated with dimethyl sulfoxide (dmso), BAPTA (20 μmol/L), or GF109203X (1 μmol/L) for 30 minutes and exposed to TxB for 60 minutes. Activations of EGFR and ERK were detected by immunoblot analysis. (E) NCM460 cells were preincubated with medium, neutralizing antibodies against EGFR (20 μg/mL), or AG1478 (.5 μmol/L) for 30 minutes and then exposed to TxB (20 nmol/L) for 1 hour. Cell extracts were incubated with C botulinum C3 exoenzyme and 6-biotin-17-NAD to adenosine diphosphate-ribosylate Rho. Proteins were loaded on the SDS-PAGE gels and immunoblotted using streptavidin conjugated with alkaline phosphatase. Cell rounding was quantitated by phase-contrast microscopy. The Western blots shown here are representative of 3 individual experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
EGFR and ERK1/2 activation are prevented by antibodies against TGF-α. NCM460 cells were preincubated with control IgG (20 μg/mL), or neutralizing antibodies against EGF (20 μg/mL), heparin-binding EGF (20 μg/mL), TGF-α (20 μg/mL), or amphiregulin (ar) (20 μg/mL) for 30 minutes and then exposed to TxB (20 nmol/L) for 1 hour. Activations of (A) EGFR and (B) ERK were detected as described in Figure 2. Results are expressed as mean ± SEM of 3 experiments per group. *P < .05, cells treated with neutralizing antibodies against TGF-α vs cells treated with control IgG in TxB-exposed groups. The Western blots shown here are representative of 3 individual experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
MMP inhibitors prevent EGFR and ERK1/2 activation induced by TxB. NCM460 cells were incubated with either vehicle DMSO or different MMP inhibitors: batimastat (BB94) (3 mg/mL) or GM6001 (25 μmol/L) for 30 minutes before the exposure of TxB (20 nmol/L). Activations of (A) EGFR and (B) ERK1/2 were detected by immunoblotting using their phosphorylation-specific antibodies. Antibodies against total EGFR and ERK1/2 were used to show equal amounts of protein were loaded into each lane. Results are expressed as mean ± SEM of 3 experiments per group. *P < .05, cells pretreated with BB94 or GM6001 vs cells pretreated with DMSO at 60 minutes in TxB-exposed groups. The Western blots shown here are representative of 3 individual experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
TxB-mediated TGF-α release is dependent on MMP activation. (A) NCM460 cells were exposed to TxB (20 nmol/L) for various periods of time. TGF-α released into the medium was measured by enzyme-linked immunosorbent assay. Results are expressed as mean ± SEM of 3 experiments per group. *P < .05, TGF-α secretion at indicated time points vs TGF-α secretion at 0 minutes with TxB stimulation. (B) Cells were preincubated with DMSO, GM6001 (25 μmol/L), or inactive GM6001 control compound (25 μmol/L) for 30 minutes before exposure with TxB for 1 or 3 hours. The concentrations of TGF-α secreted in the conditioned media were determined by enzyme-linked immunosorbent assay. Results are expressed as mean ± SEM of 3 experiments per group. *P < .05, TGF-α secretion with preincubation of GM6001 vs TGF-α secretion with DMSO in TxB-exposed groups at indicated time points. TGF-α concentrations were normalized to (A) TGF-α secretion at 0′ point and (B) 0 hours with DMSO. □, DMSO; ■, GM6001; ║, inactive GM6001.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 6
TxB-induced EGFR and ERK activation are required for IL-8 release. (A) NCM460 cells were transfected transiently with IL-8 luciferase promoter along with an internal control. Cells were incubated with TxB (20 nmol/L) at indicated time points. Cells were lysed and luciferase activities were determined. Results are expressed as mean ± SEM of 3 experiments per group. *P < .05, IL-8 expression at different time points vs IL-8 expression at 0 minutes with TxB exposure. (B) NCM460 cells were transfected transiently with IL-8 luciferase promoter. Cells were preincubated with vehicle DMSO, GM6001 (25 μmol/L), AG1478 (0.5 μmol/L), or PD98059 (10 μmol/L) for 30 minutes and exposed to TxB (20 nmol/L) for 16 hours. Cells were lysed and luciferase activities were determined. Results are expressed as mean ± SEM of 3 experiments per group. *P < .05, IL-8 expression with pretreatment with AG1478 or PD98059 vs IL-8 expression with pretreatment with DMSO in TxB-exposed groups. □, DMSO; ■, PD98059; ▨, AG1478; ▩, GM6001.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions