Fig 2: Confirmation of reproducibility of TIPRL, LC3 and CD133 as HCC biomarkers. (a) Using a different IHC cohort (Supplementary Tables 4 and 5), the levels of TIPRL, LC3, CD46 and CD133 were quantified using the ZEN program. Statistical differences (*P < 0.05; ***P < 0.0001) were calculated by a paired t-test, and % differences are shown. (n), the number of samples is indicated. (b–f) The correlations between levels of TIPRL, LC3, CD46 and CD133 were determined by the Spearman correlation equation. Single dots indicate an individual sample. (g) Diagnostic efficacies of TIPRL, LC3, CD133 and CD46 were determined by the ROC analysis. AUC, area under the curve. (h) Our study shows that the combination of TIPRL contributing to drug resistance with the cancer stem marker CD133 and autophagy marker LC3 produces novel HCC biomarkers. These biomarkers show more significant diagnostic efficacies than the CD46 currently being used as a liver cancer marker.

Fig 3: Effect of LiCl on the expression of ß-catenin, CD44, CD133 and CD44v6 proteins in adherent 2D T88 and T93 primary cell cultures. Confocal images of untreated T88 and T93 primary colon cancer cells and cells after 10 days (10 d) of LiCl incubation. Cells were dual-stained with anti-ß-catenin (red)/anti-CD44 (green) and anti-CD133 (red)/anti-CD44v6 (green) antibodies. Nuclei were counterstained with DAPI (blue).

Fig 4: Effects of FoxM1 shRNA or cDNA transduction on carcinogenicity and stemness in HCSLCs or MHCC97H cells. HCSLCs were transduced with Ad-GFP or Ad-shFoxM1. a Immunoblotting performed with anti-MnSOD and anti-FoxM1 antibodies, with ß-actin antibodies as a loading control; b and c Formed spheres and colonies (Scale bar, 200 µm); d Immunoblotting for CD133, CD44, ALDH1, Bmi1, Nanog and Oct4 in HCSLCs untreated (Mock), and transduced with Ad-GFP and Ad-shFoxM1, respectively. MHCC97H cells were transduced with Ad-GFP and Ad-FoxM1, respectively. e Immunoblotting performed with anti-MnSOD and anti-FoxM1 antibodies, with ß-actin antibodies as a loading control; f and g Formed spheres and colonies (Scale bar, 200 µm); h Immunoblotting for CD133, CD44, ALDH1, Bmi1, Nanog and Oct4in MHCC97H cells untreated (Mock), and transduced with Ad-GFP and Ad-FoxM1, respectively

Fig 5: Effects of MnSOD shRNA or cDNA transduction on carcinogenicity and stemness in HCSLCs or MHCC97H cells. HCSLCs were transduced with Ad-GFP or Ad-shMnSOD. a Immunoblotting performed with anti-MnSOD and anti-FoxM1 antibodies, with ß-actin antibodies used as a loading control; b and c Formed spheres and colonies (Scale bar, 200 µm); d Immunoblotting for CD133, CD44 and ALDH1, Bmi1, Nanog and Oct4; e The relative luciferase activity of FOXM1 promoter fragment in HCSLCs untreated (Mock), or transduced with Ad-GFP and Ad-sh MnSOD, respectively. MHCC97H cells were transduced with Ad-GFP or Ad-MnSOD. f Immunoblotting performed with anti-MnSOD and anti-FoxM1 antibodies; ß-actin antibodies were used as a loading control; g and h Formed spheres and colonies (Scale bar, 200 µm); i Immunoblotting for CD133, CD44, ALDH1, Bmi1, Nanog and Oct4; j The relative luciferase activity of FOXM1 promoter fragment in MHCC97H cells untreated (Mock), and transduced with Ad-GFP and Ad-MnSOD, respectively