Figure 7.

NANOG is regulated by HH-GLI signalling and NANOG function is epistatic to an active HH-GLI pathway. (A) Gene expression changes after enhancement (through PTCH1 kd or GLI1 expression) or repression (through SMOH kd) of HH-GLI signalling in GBM-8, -12 and U87 cells. Values are RT–qPCR ratios over controls after normalization at 4 days after transduction. (B) Western blot analysis of NANOG protein using KAb in U87 cells transduced with LV-GFPcontrol-1, LV-GLI1, LV-shSMOH or LV-GLI3R. HSP90 is shown as loading control. NANOG protein is greatly diminished in cells with compromised HH-GLI signalling. (C) Representative images of dissected whole brains with developed ‘red/green' brain tumours after orthotopic xenotransplantation of GBM-8 gliomaspheres transduced with control red (RFP⁺) plus green (GFP⁺) lentivectors expressing different shRNAs as indicated. Each row shows the images of dorsal brains with anterior to the left under visible, green or red fluorescence. Green/red FACS ratios are given in the right column. (D) Quantification and evolution of FACS ratios in red/green competition assays in vivo after normalization with controls, which are equated to 1. The number of mice analysed at each passage (P) is also given (n) for each case. Scale bar, 3.5 mm (C).