Fig. 2

SOX2 plays an important role in colorectal metastasis. a Hierarchical clustering heatmaps of the 33 significantly upregulated genes in 13L organoids. b qRT-PCR analysis of SOX2 in paired organoid lines. Values were normalized to mean levels in 13a organoids. Error bars indicate SEMs. **p = 0.0050, ****p < 0.0001 (one-way ANOVA). c Western blot analysis of the SOX2 protein expression in paired organoid lines. α-tubulin was used as a loading control. d Representative IHC sections for SOX2 in human colorectal tumor tissues and organoids. The scale bar represents 100 μm. e Representative micrographs of Dox-transduced the 13 L-shRNA-1/2 organoids after 5 days and stained with phalloidin–F-actin. The scale bar represents 100 μm. f Representative micrographs of colonies arising from the 13L-shRNA-1/2 organoids (top), with magnified insets showing colonies (bottom). The scale bar represents 200 μm. Colony forming efficiency in f was calculated and compared (right). Error bars indicate SEMs. **p = 0.0078, (two-way ANOVA). g Proliferation of the 13L-shRNA-1/2 organoids were examined by CTG cell viability assays following 3- or 5-days growth in the presence or absence of Dox. Error bars indicate SEMs. **p = 0.0019, ****p < 0.0001 (two-way ANOVA). h Representative macroscopic photographs (top) and histology sections (H&E, bottom) of the livers of NOD mice transplanted with the 13L-shRNA-1/2 organoids. Arrowheads, metastatic foci. n = 5 mice per group. Scale bars, 1 cm (top) and 4 mm (bottom). i Representative SOX2 immunostaining of liver metastases of the 13L-shRNA-1/2 organoids. Scale bars, 200 μm (top) and 100 μm (bottom)