Fig 1: Hypoxia increases the self-renewal capacity of patient-derived TIC cultures. (A) HIF1A protein expression under normoxic (N) and hypoxic (H) culturing conditions over 3 d (72 h) and 7 d in T6 TICs. TUBA staining was used as a loading control and HepG2 cells exposed to hypoxia for 24 h were used as a positive control (+). (B) Representative image of patient T6-derived TICs, cultured under normoxia (N) and hypoxia (H). Scale: 100 µm. (C-H) Self-renewal capacity as determined by the 1000 cell sphere formation assay (C-E) in T6 (C), T18 (D) and T20 (E) cultures and by the single cell assay (F-G) in T6 (F) and T18 (G) cultures. Sphere formation was observed over several passages (P). (H) Self-renewal capacity was determined by a limiting dilution assay at multiple cell doses under normoxia and hypoxia. Results from one experiment using T6 TICs are shown and ELDA was used to assess significance. C-G; Data are representative of at least three independent experiments. (I-J) Hypoxia induces the expression of stem cell markers. (I) Flow cytometry staining of POU5F1 in T18 TICs under normoxic and hypoxic conditions and quantification of mean fluorescence intensity (MFI) of POU5F1 in two independent experiments (data normalized to normoxia). (J) Flow cytometry staining of ALDH1A1 in T18 TICs under normoxic and hypoxic conditions and quantification of mean fluorescence intensity (MFI) of ALDH1A1 in two independent experiments (data normalized to normoxia). Data are presented as mean ± SD except for F, G, and H, which are presented as a mean with 95% confidence interval, *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant.