Fig 2: Exogenous SHH ligands can active Hh signalling in CAFs(A) Flow cytometric analysis of marker proteins in CAFs and NOFs demonstrating that CAFs are positive for α-SMA and vimentin but negative for cytokeratin 8, whereas NOFs are positive for vimentin but negative for α-SMA and cytokeratin 8. (B) Representative images of morphology of passage 3 CAFs and NOFs. (C) Representative fluorescent images of PTCH- (red), α-SMA- (green) and DAPI-labelled (blue) CAFs and NOFs. (D) Western blot analysis of Gli-1 and PTCH in CAFs and NOFs after 48 h treatment with rSHH (1 μg/ml). GAPDH served as the loading control. (E) Western blot analysis of Gli-1 and PTCH in CAFs in the absence or presence of rSHH (1 μg/ml) or Cyclopamine (10 μM) for 48 h.

Fig 3: RUNX1 decreased 5-FU sensitivity of CRC cells via Hedgehog signaling pathway. A. 5-FU sensitivity measured by CCK8 assays in HCT116/RUNX1, SW480/shRUNX1, HCT116/Vector and SW480/Scramble cells with the different concentrations of 5-FU treated; error bars, SD. B. Apoptosis rate measured by flow cytometer in HCT116/RUNX1, SW480/shRUNX1, HCT116/Vector and SW480/Scramble cells with 5-FU treated; error bars, SD. C. Expression of RUNX1 in HCT116, SW480 and RKO cell lines with 5-FU treated. D. Expression of ABCG2 in HCT116/RUNX1, SW480/shRUNX1, HCT116/Vector and SW480/Scramble groups. E. Expression levels of GLI1 PTCH1 ABCG2 mRNA in HCT116/Vector and HCT116/RUNX1, SW480/scramble and SW480/shRUNX1 group; error bars, SD. F. HCT116 and RKO cell proliferation determined by CCK8 assay in Vector and RUNX1, vector with GDC and RUNX1 with GDC groups G. Expression of ABCG2 and targeted genes activated by Hedgehog signaling pathway in HCT116/Vector, HCT116/RUNX1, HCT116/RUNX1 with GDC groups.

Fig 4: The active IHh signal pathway can be attenuated by siGli1 but not by cyclopamine. Cells s were treated with Gli1 siRNA and cyclopamine (10 μM) for 24 h. Changes in the expression of IHh, PTHrP, PTCH, SMO, and Gli1 were examined by qPCR (a) and WB (b). The data are presented as mean±S.D. of three independent experiments (*P<0.05, **P<0.01, #P>0.05 versus controls)

Fig 5: Expression of GLI1 and p-AKT in AML cell lines.(a,b) The transcript expression of GLI1 in both wild-type and GLI1 overexpressing HL60 cells (a) and NB4 cells (b) treated with 20 μM GANT61 or 20 μM LY294002. (c,d) The protein expression of GLI1 and p-AKT in both wild-type and GLI1 overexpressing HL60 cells (c) and NB4 cells (d) treated with 20 μM GANT61 or 20 μM LY294002. *P < 0.05, **P < 0.01.