Figure 4.

Suppression of endogenous galectin‐3 impairs the interleukin‐1β (IL‐1β) response in a p38 mitogen‐activated protein kinase (MAPK) ‐independent manner. (a) Stratified human corneal keratinocytes were transfected with galectin‐3 small interfering RNA (siRNA) (Gal3KD) or control siRNA (Scr) using Lipofectamine 2000. The cultures were then incubated with IL‐1β (10 ng/ml) for 0, 1, 2 and 6 hr at 37°. Levels of IL‐8 and IL‐6 in the cell culture media, and galectin‐3 in cell lysates, were determined by immunoblot. (b) Real‐time quantitative PCR analysis of IL‐8 and IL‐6 gene expression in stratified human corneal keratinocytes transfected with galectin‐3 or control siRNA. The cultures were incubated with IL‐1β (10 ng/ml) for 0, 1, 2 and 6 hr at 37°. (c) Cells were treated with IL‐1β (10 ng/ml) for 0, 5, 15 and 30 min at 37°. Phosphorylated p38 MAPK (pp38) and total p38 MAPK (p38) were measured by immunoblot. (d) Cells transfected with galectin‐3 or control siRNA were incubated with or without 10 ng/ml IL‐1β for 6 hr at 37°. Levels of IL1R1 in cell lysates were determined by immunoblot. Cell surface labelling of IL1R1 and galectin‐3 was carried out using biotinylation as described in Materials and methods. Results in (a) and (b) represent four independent experiments and data are reported as mean ± standard deviation. Significance was determined using two‐way analysis of variance with Sidak's post hoc multiple comparison test. ****P < 0·0001; ns, non‐significant.