Fig. 3: The liver CSC signatures were enhanced in vivo.

a Representative images of tumour and H&E staining for first, second and third primary orthotopic injection tumour sections. Scale bars represent 64 μm. b RT-qPCR analysis of AFP, GPC3 and CK19 were examined and liver CSC markers such as CD133, CD44 and CD24 for the three malignant tumour tissues were compared to those in normal mice liver. Gene expression levels were normalised to those of GAPDH (*p < 0.01, **p < 0.001, ***p < 0.0001, ****p < 0.00001). c Immunohistochemical staining of formalin-fixed paraffin-embedded liver tumour tissue derived from serial transplantation (miPS-Huh7cm cells) and histogram showing area of immunopositive cells of each marker. Samples representative of each tumour were stained with antibodies recognising AFP, GPC3 and CK19. Also, antibodies recognising liver CSC like CD44, CD24 and CD133 were applied to the tumour tissues. All data were from three independent experiments (n = 3). Scale bars represent 64 μm. d FACS analysis shows AFP⁺/CD44⁺, GPC3⁺/CD44⁺ and CEA⁺/CD44⁺ subpopulations in the tumour single-cell suspension.