FIG 4.

TGF-β1 protects C. difficile TcdA-treated IEC-6 cells from apoptosis and necrosis. IEC-6 cells were incubated for 24 h in culture medium (control) with TcdA (10 ng/ml), TGF-β1 (10 ng/ml), or TcdA plus TGF-β1 (10 ng/ml) and stained with FITC-conjugated annexin V and propidium iodide (PI), followed by flow cytometry analysis. Density plots with PI versus annexin V-FITC show that viable cells have low annexin V-FITC and low PI staining (lower left quadrant), apoptotic cells have high annexin V-FITC and low PI staining (lower right quadrant), and necrotic cells have high PI and annexin V-FITC staining (upper quadrant). The numbers of apoptotic and necrotic cells were increased in the presence of TcdA compared to those in the control (A, C, E, and F). Concomitant addition of TGF-β1 partially rescued cells from apoptosis (D and E) and totally rescued cells from necrosis (D and F). The addition of TGF-β1 alone did not affect these events (B, E, and F). *, P < 0.05 (ANOVA and Bonferroni's test).