Fig. 4
NAC and GSH enhanced tumorigenesis and promoted liver cancer stem cell markers and stemness genes. a, b Limiting dilution assay showing the effects of NAC and GSH on tumorigenicity in nude mice. 2 × 103, 2 × 104 and 2 × 105 MHCC-97L cells were injected subcutaneously into nude mice. The tumor incidence rate for each group was recorded at the end of the experiments after 4 weeks. c Tumor initiating capacity was analyzed by the confidence intervals (CIs) with the formula of CI = 1/(stem cell frequency). d mRNA expression of stemness-related genes upon NAC and GSH treatment in HCC cells. e–f Schematic diagram illustrating the workflow of investigating the expression of CSC markers and stemness-related genes in HCC tumors harvested from mice in the orthotopic liver injection model using MHCC-97L cells. Detection of mRNA expression of stemness-related genes in tumors by qPCR with control, low- and high-dose NAC/GSH treatment. Data are expressed as mean ± SD from three independent experiments. *P < 0.05, ** P < 0.01 and ***P < 0.001 vs. control; one-way ANOVA followed by Dunnett comparison test