Fig. 1: Areg signaling is altered but not abrogated in the context of HS inhibition.

A Diagram of different types of heparan sulfate (HS)-presenting proteoglycans, heterogeneity in sulfation along a single HS chain, and epidermal growth factor receptor (EGFR) ligand interactions with HS. ECM extracellular matrix, GlcNAc N-acetylglucosamine, GlcA glucuronic acid, GlcNS(6S) N-sulfoglucosamine (6-O-sulfated), IdoA(2S) iduronic acid (2-O-sulfated), AREG amphiregulin, EGF epidermal growth factor. B Western blotting for phospho-EGFR (Y1068), phospho-AKT (S473), phospho-ERK (T202/Y204), and β-actin of vehicle or sodium chlorate (NaClO₃)-treated (16–18 h) LLC cells, stimulated (15 min.) with vehicle, mouse AREG (500 ng/ml), or mouse EGF (100 ng/ml). Representative western blots shown. n = 3 per condition, graphs contain all values from three experiments. C Flow cytometry using HS-directed antibodies 10E4 or JM403 on LLC cells treated with vehicle (n = 3) or NaClO₃ (n = 4) (16–18 h). Representative flow cytometry plots shown. Gating based on fluorescence-minus-one (FMO) controls. Percent staining positive displayed in plots. Graphs contain all values from two experiments. D Western blotting for phospho-AKT (S473), phospho-ERK (T202/Y204), and β-actin of vehicle or mouse AREG-stimulated (at various concentrations) (15 min.) Ba/F3-Egfr cells. Representative western blots shown. n = 3 per condition, graphs contain all values from three experiments. E Western blotting for phospho-EGFR (Y1068), phospho-AKT (S473), phospho-ERK (T202/Y204), and β-actin of vehicle or NaClO₃-treated (16–18 h) A549 cells, stimulated (15 min.) with vehicle, human AREG (500 ng/ml), or human EGF (100 ng/ml). Representative western blots shown. n = 3 per condition, graphs contain all values from three experiments. F Western blotting for phospho-EGFR (Y1068), phospho-AKT (S473) phospho-ERK (T202/Y204), and β-actin of vehicle or NaClO₃-treated (16–18 h) Col14-LMC, stimulated (15 min.) with vehicle, mouse AREG (500 ng/ml), or EGF (100 ng/ml). Col14-LMC were gated/sorted as shown in Supplementary Fig. 2 (negative bead enriched), then at ~24 h post-plating non-adherent cells were washed away/media changed, with subsequent vehicle/NaClO₃ treatment. Representative western blots shown. n = 4 per condition, graphs contain all values from four experiments. Statistical analysis done for western blot/flow cytometry data where two groups were compared using two-tailed unpaired t-tests, and for western blot data where four groups were compared using one-way ANOVA. Mean and standard error displayed on graphs; ns not significant, *0.01 <p < 0.05, **0.001 <p < 0.01, ****p < 0.0001. Source data are provided as a Source Data file.