Gli1-GFP reporter assays

Cells (3–5 × 10⁴) plated in 24-well plates in different CM and/or drug conditions were simultaneously transfected in different wells with either 7-Gli1-GFP or m-Gli1-GFP³⁹ reporters. Cells in each condition were also transfected with a constitutively expressing GFP-Ctrl vector on the same vector background in a separate well. Forty-eight hours post transfection, the number of GFP⁺ cells in each condition (indicating activated GLI signalling) was counted using a fluorescence microscope and normalized to the number of GFP⁺ cells in the well containing GFP-Ctrl vector to account for differences in transfection efficiencies of cells cultured in different CM and/or drugs. Results from three or more independent experiments using different sets of CM were grouped and graphed as % of 7-Gli1 or m-Gli1 GFP⁺ cells. Further details on the reporters will be described elsewhere.