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. 2016 Oct 25;11(10):e0162878. doi: 10.1371/journal.pone.0162878

Fig 2. NHBA binds epithelial cells via direct interaction with heparan sulfate proteoglycans.

Fig 2

A) Hec-1B epithelial cells were pre-treated with heparinase III or chondroitinase ABC and then used for binding assay with the wild-type NHBA protein. The panels show untreated cells (left) and cells treated (right) with heparinase III (upper panel) or with chondroitinase ABC (lower panel). Cells were stained with a mouse monoclonal anti-heparan sulfate or anti-chondroitin sulfate antibody, respectively, and a secondary fluorescent antibody (red staining). NHBA binding was detected with a primary polyclonal rabbit anti-NHBA serum and a secondary fluorescent antibody (green staining). B) Fluorescence of Hec-1B cells was measured after pre-treatment with heparinase III or choindroitinase ABC and incubation of NHBA, using Tecan fluorescent plate reader. Heparan-sulfate, choindroitin sulfate and NHBA were detected with the same antibodies used for confocal microscopy analysis. Results are reported as percentage of fluorescence with respect to the untreated cells (set to 100%). The graph shows the results of one representative experiment performed in triplicate. Each bar represents the mean number of fluorescence units, error bars indicate the standard deviation (n = 3). NS not significant; ** p value <0.01; *** p value < 0.001; **** p value < 0.0001 (Unpaired t-test with Welch’s correction). C) NHBA protein was untreated or pre-incubated with increasing concentrations of heparin (0.01, 0.1, 1 μg/ml), and then used for binding assays with Hec-1B cells. Cells were then stained with a primary mouse polyclonal anti-NHBA serum and a secondary fluorescent antibody. Mean fluorescence intensity (Fluorescence) units, measured with a Tecan reader, are reported on the vertical axis. The graph shows the results of one representative experiment performed in triplicate. Each bar represents the mean number of fluorescence units, error bars indicate the standard deviation (n = 3). **** p value < 0.0001 (ANOVA test). D) Binding of the wild-type NHBA protein to wild-type CHO-K1 or CHO pgsA-745 epithelial cell monolayers and co-localization with heparan sulfate. NHBA (green staining) and heparan sulfate (red staining) were detected as in panel A. Scale bars represent 20 μm.