FIGURE 1.

A, representative confocal micrographs show HβD2 expression in confluent cultures of HT-29 cells that were treated with medium alone or containing HA-35 (1 μm) for 12 h. Cells were fluorescently immunostained for HβD2 protein (red), and nuclei were stained with DAPI (blue). NS, immunostaining control in which no α-HβD2 antibody was utilized. B, mean secreted HβD2 as measured by ELISA in the culture medium of HT-29 cells treated for 9 h with medium alone or containing HA-35 (10 μm). No significant difference in secreted HβD2 peptide was detected between media or HA-treated cultures. C, average densitometric quantification of immunoblots from four individual experiments in which the abundance of HβD2 protein relative to GAPDH protein was evaluated in whole cell lysates of HT-29 cells. Replicate cultures were treated with HA-35 (1 μm) at 3-h time intervals (0–24 h) in each experiment. Significance of differences in normalized HβD2 expression was evaluated by comparison of each time point with control treatment using Student's t test (***, p < 0.001). D, representative Western blots demonstrating HβD2 protein expression relative to GAPDH in the whole cell lysates of HT-29 cells treated with medium alone, medium containing HA-35 (10 μm), and medium containing HA-35 (10 μm) that was predigested with Streptococcus dysgalactiae hyaluronidase at 0.025 unit/ml for 16 h at 37 °C. Error bars, S.E.