Fig 2: Clustering of 17 HCC cell lines with protein expression and phosphorylation. (A) GMM clustering of the cell lines based on the RPPA analysis of EpCAM and CD90. A VEV model (ellipsoidal, equal shape) with three components (clusters) was selected using BIC. The clusters are colored by red (CD90+), blue (EpCAM+) and grey (Neutral). Each point is the mean of six replicates. Error bars represent the SDs. Error ellipses denote the 1 s regions of mixture Gaussian components. (B) Heat map and hierarchical clustering of the cell lines and all the RPPA analysis. Average linkage and Euclidean distance were used to create the dendrograms. The clustering result is depicted by colored bars (dark blue, Cluster 1; magenta, Cluster 2). The GMM clusters are also depicted by bars with the same color as (A).

Fig 3: The kinetics of carcinogenesis induced by SPTBN1 heterozygous deletion were accelerated by cotreatment with DDC.A, Liver images of WT and Sptbn1+/-mice cotreated with DDC. Arrows point to HCC in DDC-treated Sptbn1+/– mice. Six- to eight-week-old male WT and Sptbn1+/- mice (n = 4 for each experiment with 3 experimental replicates) were fed a diet containing 0.1% DDC for 5 months. B, H&E staining of liver sections from WT and Sptbn1+/- mice induced by DDC. White bars represent 200 µm. C, Tumor incidence rate (n = 8). D, H&E staining of liver tissues from WT and Sptbn1+/- mice aged 6 to 8 weeks. The development and architecture of liver tissues are normal in Sptbn1+/- mice. White bars represent 100 µm. E, The ratio of liver weight to body weight. The liver and body weight of WT and Sptbn1+/- mice was measured 1 month after feeding a 0.1% DDC-containing diet (n = 4; **P < .01 vs WT). F, The morphology of cultured HSCs in 3, 7, 14, and 21 days. G, The number of EPCAM-positive cells in HSCs was quantitatively evaluated by flowcytometric analysis. H, The stemness nature of cultured HSCs were examined by RT-PCR following by DNA gel electrophoresis. I-J, mRNA and protein levels of EPCAM in HSCs of Sptbn1+/- mice detected by qRT-PCR (I) and Western blot (J). Data is representative of 3 independent experiments. Significance of the difference was evaluated using the Student t test (*P < .05; **P < .01 vs WT). K, Immunofluorescence staining. SPTBN1 heterozygous deletion enhanced expansion of EPCAM-positive (arrow) HSCs in DDC-treated livers. CV, Central veins; PV, portal veins. White bars represent 100 µm. Number of EPCAM positive cells were counted (n = 4, mean ± standard deviation). Significance of the difference was evaluated using the Student t test (**P < .01 vs WT).

Fig 4: SPTBN1 inhibited malignant transformation by promoting autophagy and the Hippo pathway in murine HSCs.A and B, The effect of RAPA was analyzed by qRT-PCR and Western blot. A, The mRNA levels of autophagy-related genes Becn1 and Atg4b, EMT-related genes Vim and Cdh1, Afp and Epcam were reversed by RAPA in HSCs from DDC-treated Sptbn1+/- mice (n = 3; *P < .05, **P < .01 vs WT, #P < .05 vs Sptbn1+/-). B, The decreased ratio of LC3B-II/I in HSCs from DDC-treated Sptbn1+/- mice was rescued by RAPA (n = 3; **P < .01 vs WT, ##P < .01 vs Sptbn1+/-). C, H&E staining of liver sections and immunofluorescence staining of EPCAM/SQSTM1 and VIM/CK19. Upper, H&E staining showed the morphological differences of liver tissues in DCC-treated Sptbn1+/-mice with or without RAPA injection. Black bars represent 100 µm. Lower, Immunofluorescence staining of EPCAM/SQSTM1 and VIM/CK19. The number of EPCAM/SQSTM1 double-stained cells (yellow arrow pointed) and VIM/CK19 double-stained cells (white arrow pointed) was decreased in the livers of RAPA- and DDC-treated Sptbn1+/- mice compared with DDC-treated Sptbn1+/- mice. White bars represent 50 µm. D, Kyoto Encyclopedia of Genes and Genomes enrichment analysis of EPCAM+ HSCs from WT and Sptbn1+/- mice. Forty-four different genes in Hippo-YAP signaling pathway were enriched. E, The effect of SPTBN1 on the Hippo-YAP pathway in HSCs was examined by Western blot. Protein levels of LATS1 and YAP were increased, whereas those of p-LATS1 and p-YAPS127 were decreased in HSCs from DDC-treated Sptbn1+/- mice (n = 3; **P < .01 vs WT). F. Immunofluorescence staining of EPCAM/YAP. EPCAM/YAP double-stained cells (arrow) were increased in the livers of DDC-treated Sptbn1+/- mice. White bars represent 50 µm. G, Western blot. The protein level of p-LATS1, LATS1, and YAP was analyzed by Western blot in the MEF cells (*P < .05, **P < .01 vs MEF+/+).

Fig 5: Bufalin downregulates the expression levels of CD24 and ESA. In the subcutaneous xenograft model, the control and bufalin-treated tumors were excised and analyzed using immunohistochemistry and/or immunofluorescence to measure the expression levels of CSC-associated markers. (A) Immunohistochemical staining with anti-ESA was performed using sections of subcutaneous xenograft; the positive rate of the CSC-associated marker, ESA, was determined based on the immunohistochemistry. Data are expressed as the mean + standard deviation (n=6). (B) Expression levels of CD24 was assessed using immunofluorescence. CSC, cancer stem cell; ESA, epithelial specific antigen.