Additional Supporting Information may be found in the online version of this article.

HEP_24739_sm_SuppFig1.tif4523KSupporting Information Fig. S1. Representative flow cytometry histogram plot showing the distribution of CD133-expressing cells in unsorted and sorted HCC cells (Huh7 and PLC8024). The red histogram represents the isotype control. CD133 expression of unsorted (black) and sorted CD133+ (green) and CD133- (blue) cells is relative to this isotype control. The purity for the positive fraction ranged from 90-98%, while the negative fraction achieved greater than 97% purity.
HEP_24739_sm_SuppFig2.tif1127KSupporting Information Fig. S2. Immunoblot analyses of the expression of IL-8 receptors (CXCR1 and CXCR2) and NTS receptors (NTSR1 and NTSR2) in a panel of liver cell lines.
HEP_24739_sm_SuppFig3.tif4012KSupporting Information Fig. S3. Compared to non-transduced cells, Huh7 cells with IL-8 suppression displayed reduced engraftment ability, as demonstrated by significantly decreased tumor size (Mann-Whitney test: p = 0.05) and decreased tumor incidence (n = 5 per group).
HEP_24739_sm_SuppFig4.tif4556KSupporting Information Fig. S4. Compared to non-transduced cells, Huh7 cells with IL-8 suppression (clones 30 and 31) generated fewer spheroids that could not be serially propagated (ANOVA: p < 0.001; Multiple comparison with Bonferroni correction: *p < 0.001; **p < 0.001).
HEP_24739_sm_SuppFig5.tif3626KSupporting Information Fig. S5. (A) Increased expression of NTS and CXCL1 in CD133+ liver TICs as compared to CD133- cells in freshly resected clinical HCC specimens (t-test: *p = 0.002; **p = 0.003; ***p = 0.005; ****p = 0.03). (B) qPCR analyses of CD133, IL-8 and CXCL1 expression in sorted CD133 subpopulations (Huh7) in the presence or absence of exogenous NTS (1 µg/mL) or IL-8 (100 ng/mL) for 0-, 1- or 2-hr time points. (C) qPCR analyses of IL-8 and CXCL1 expression in CD133-expressing HCC cell line Hep3B in the presence or absence of exogenous NTS (1 µg/mL) or IL-8 (100 ng/mL) for 0-, 1- or 2-hr time points. (D) A reduction in IL-8 and CXCL1 expression was detected in IL-8-repressed PLC8024 cells (clones 30 and 31) when compared to non-transduced cells (ANOVA, p = 0.002; Multiple comparison with Bonferroni correction, p = 0.009).
HEP_24739_sm_SuppFig6.tif7375KSupporting Information Fig. S6. Significant reduction of CD133 expression in CD133 shRNA knockdown clones 44 and 47, compared with non-transduced cells, and in PLC8024 CD133+ liver TICs, as detected by flow cytometry.
HEP_24739_sm_SuppFig7.tif4213KSupporting Information Fig. S7. Dual-color flow cytometry analyses of CD133 with CD90, EpCAM and CD44 in HCC cell lines (Huh7, PLC8024, Hep3B) and HCC clinical specimen (Patient 72).
HEP_24739_sm_SuppInfo.doc38KSupporting Information Table S1. Primer sequences for qPCR analysis.
HEP_24739_sm_SuppTab1.doc37KSupporting Information Table S2. List of commonly dysregulated genes with a relative fold change greater than 2 in CD133+ vs. CD133- cells in the HCC cell lines Huh7 and PLC8024 (MAS5 and RMA).
HEP_24739_sm_SuppTab2.doc217KSupporting Information Table S3. Validation of commonly dysregulated known hepatic stem cells / TIC markers and genes involved in the IL-8 pathway in sorted CD133 subpopulations (Huh7 and PLC8024) by qPCR analysis.
HEP_24739_sm_SuppTab3.doc39KSupporting Information

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