Fig. 2: ATO attenuates liver CSC-associated traits in HCC cells.

a The effects of ATO, sorafenib, 5-FU, and doxorubicin on tumorsphere formation. All the reagents were added into the tumorsphere system at day 5. Tumorsphere formation (n = 3) was quantified as the total cross-sectional areas of tumorspheres/number of tumorsphere per group (area average) at day 8. Bars, 500 μm. *P < 0.05, NS: not significant, determined by two-sided Student’s t-test. b, c The effects of ATO on CD13⁺/CD133⁺/EpCAM⁺ subpopulation percentage (b) and tumorsphere formation of HCC cells (c) at indicated concentrations (μM). Bars, 200 μm. Tumorsphere formation (n = 3) was quantified as the total cross-sectional areas of tumorspheres per group (area sum) (similarly hereinafter if not mentioned). “*” or “**” indicate the comparison for the “area sum” of tumorspheres with the control (0 μM) in the bar graphs. d Relative values of “stemness” gene and HCC marker gene expression in HCC cells. e Immunofluorescence analysis of AFP expressions after ATO treatment in HCC cells. Bars, 20 μm. f–h Comparison of tumorsphere formation (n = 3, f), relative values of “stemness” gene and HCC marker gene expression (by qRT-PCR, g), and chemoresistance (h) from Huh7.5.1 cells-derived CD13⁺/CD133⁺/EpCAM⁺ subpopulation treated with ATO or PBS. Bars, 500 μm. *P < 0.05, **P < 0.01, determined by two-sided Student’s t-test