Fig. 2. Galectin-3 stimulates reciprocal signaling and influences epithelial MMP9 secretion.

(A) Fluorescence intensity measured in human corneal epithelial cells carrying the MMP9 reporter construct (EpiGFP). The serum-free conditioned media (CM) of fibroblasts grown alone or in coculture with epithelial cells for 24 h were diluted in a 1:1 ratio with fresh media and used to stimulate homotypic cultures of EpiGFP cells for an additional 24 h. n = 3 independent experiments. (B) Exogenous soluble galectin-3 (rhGal3) or BSA in serum-free media were incubated with fibroblasts for 24 h. The conditioned media were pre-cleared with α-lactose agarose beads to remove galectin-3 prior to incubation with epithelial cells. After an additional 24 h, the epithelial cell culture supernatants were analyzed by gelatin zymography to determine the abundance of MMP9. The presence of galectin-3 in pre-cleared conditioned media was evaluated by immunoblotting. n = 4 independent experiments. (C) Epithelial cells were transfected with scrambled (siScr) or galectin-3 (siGal3) siRNA, and the presence of galectin-3 in epithelial cell lysates was assessed by immunoblotting. After 48h of transfection, cells were incubated with fibroblasts in a mixed coculture system for an additional 24 h. MMP9 abundance in the media was quantified by gelatin zymography and shown as a scatter plot. n = 7 independent experiments. (D) Same as (C) except that galectin-3 was knocked down in fibroblasts (siGal3) that were then cocultured with epithelial cells. n = 4 independent experiments. The data represent the mean ± S.D. Significance was determined using one-way ANOVA with Tukey’s post hoc test (A, B) or the Wilcoxon test (C, D). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.