Fig 2: Regulation of epithelial-mesenchymal transition (EMT) genes by the Shh signalling pathway. (A) The mSKP spheres after CP or Pur treatment were stained with a-SMA or N-cadherin (N-cad; green), and with F-actin (red). DAPI (blue) indicates nuclei. (B) The mRNA expression of a-Sma, Cdh2 (N-cad), vimentin (Vim), fibronectin (Fn1) and Tgf-ß1 was measured by qPCR in CP-treated mSKPs (with or without Pur) at 72 h. (C) The relative protein levels of a-SMA, N-cad and fibronectin were analysed by Western blot analysis. The housekeeping protein ß-actin was used to control for loading. *P < 0.05. Scale bars: 50 µm

Fig 3: The effect of purmorphamine (Pur) on mSKPs. (A) mSKP spheres formed at passage 0 (P0), P1 or P2 after Pur treatment. (B) The cell proliferation of mSKPs after Pur treatment was examined by WST-1 assay at P1 and P2. (C) The sphere-forming efficiency of mSKPs was measured. The number of spheres with a diameter more than (a) 20 µm or (b) 50 µm was compared between the control and Pur-treated groups. (c) The relative abundance of large (>50 µm) and small (>20 µm) spheres was measured at P1 and P2. (D) The mRNA expression of Oct4, Nanog, c-Myc, Klf4, N-cad, Twist, Ngfr, Smo, nestin, Lsd1, Bmi1, Sox9, Patched1 and Gli1 was measured by qPCR in mSKPs. Values were normalized against GAPDH and depicted as fold-change values relative to the control (no Pur treatment; control value = 1). *P < 0.05. Scale bars: 100 µm