Fig. 5: Engineered GPC3 inhibits Shh signaling.

a Fluorescence detection of GPC3 core protein and HS oligosaccharide chains in NIH3T3 cells. Cells transfected with engineered GPC3 (3,4) or negative controls (pCMV6XL4) (1,2) were incubated in the presence (2,4) or absence (1,3) of bioHaHS and were stained with a GPC3 antibody (GPC3) or TRITC-conjugated streptavidin. b Cells expressing engineered GPC3 were incubated with or without biotinylated HS, and FRET was performed. c NIH3T3 cells were transfected with GPC3-O or vector control (pCMV6XL4), and incubated with different types of GAG chains. A Hh luciferase reporter assay was then performed. Bars represent the fold stimulation induced by Shh. d Shh binds to GPC3-O-bioHS in the context of intact cells. 1, Wild-type GPC3 with Shh CM; 2, GPC3-O with Shh CM; 3, GPC3-O-bioHS with control CM; 4, GPC3-O-bioHS with Shh CM. e Relative fluorescence intensity of Shh bound to the surface of NIH3T3 cells expressing wild-type (wt) or engineered GPC3. wt GPC3, wild-type GPC3. All experiments were repeated three times with similar results. Error bars represent means of triplicates ± SD. p-values in all cases are compared with the corresponding negative control pCMV6XL4. Scale bars: 20 μm. Source data are provided in a Source Data file.