Hh expression is maintained in primary cell lines and can activate reporter cells in a gradient A) The fibroblast Hh pathway reporter cell line GGM was cocultured with indicated primary cell lines for 3 days. Representative fluorescence and brightfield images show GFP expression in reporter cells adjacent to cancer cells. Administration of Hh inhibitor KAAD-cyclopamine (100 nM) strongly reduced pathway activation. B) Quantification of GFP positive GGMs following coculture by flow cytometry. ns, not significant; *** p < 0.001. Percentage of GFP positive GGMs in the absence of tumor cells was 0.135 ± 0.05%, and 0.141 ± 0.06% in the presence of KAAD-cyclopamine. C) qPCR of SHH transcript in shCTR and shSHH PC053M cells relative to GAPDH. D) PC053M knockdown cells stained with anti-HH antibody 5E1(red) or isotype control (grey). Representative histogram overlay of flow cytometry experiment is shown. E) GGM coculture with knockdown cells imaged after 3 days. F) Quantification of GFP positive GGMs after coculture by flow cytometry shows strong reduction of paracrine pathway activation after knockdown of SHH in PC053M cells. *** p < 0.001.