Fig. 2

CD44-dependent interaction of recombinant human proteoglycan-4 (rhPRG4) with HEK Blue-TGF-β cells and associated modulation of phosphorylated Smad3 (pSmad3) and TGF-β/Smad signaling pathway. HEK Blue-TGF-β is an engineered cell line produced by transfecting human embryonic kidney (HEK) cells with TGF-β receptor 1 (TGF-β R1), Smad3, and Smad4 genes. Activation of TGF-β R1 results in expression of secreted alkaline phosphatase (SEAP) whose activity can be determined colorimetrically. Rhodamine-labeled rhPRG4 was incubated with HEK Blue-TGF-β cells ± anti-CD44 or isotype control (IC) antibodies and corrected total cell fluorescence (CTCF) was determined. pSmad3 immunocytostaining was performed using an antibody against pSmad3 and pSmad3 fluorescence intensity was determined. Activity of TGF-β/Smad pathway was determined colorimetrically. *p < 0.001; **p < 0.01; n.s., non-significant. Scale in a = 40 μm; scale in c = 50 μm. a Representative images showing rhPRG4 internalization (white arrows) by HEK-TGF-β cells and co-localization with CD44 receptor (white arrows in merged image). b rhPRG4 uptake by HEK-TGF-β cells was reduced by CD44 receptor neutralization. c Representative images showing pSmad3 staining in HEK-TGF-β cells following TGF-β stimulation (white arrows) ± rhPRG4 treatment. d pSmad3 immunocytostaining was reduced following rhPRG4 treatment. e rhPRG4 treatment dose-dependently reduced TGF-β/Smad signaling pathway in TGF-β-stimulated HEK-TGF-β cells