Figure 3. IKKβ deletion in initiated cells enhances proliferation and self-renewal of HCC progenitors.

(A) DEN-induced HCC (dih) cell strains from Ikkβ^f/f mice were cultured and infected with Adv-GFP or Adv-Cre. Whole cell lysates of infected dih10 cells were immunoblotted for IKKβ (bottom panel). The cells were further cultured without serum on Petri dishes to form hepatospheres (upper panels). Numbers of 1⁰ and 2⁰ hepatospheres formed per 100 plated cells were determined (n = 3; *: p < 0.05). Magnification bar = 100 μm.

(B) Ikkβ^f/f dih10 cells were infected with an empty retrovirus (vector) or a retrovirus expressing IκBα super-repressor (SR). Stably transfected cells were selected and cultured as above and hepatosphere formation was analyzed (n = 3; *: p < 0.05).

(C, D) IKKβ expressing (Ikkβ^f/f) and deficient (Ikkβ^Δ) dih10 cells (2.5 × 10⁶ each)were s.c. injected into 8 weeks old C57BL/6 mice. (C) Allograft volume was measured weekly (n = 5; *: p < 0.01). Dissected tumors are shown on the right. (D) BrdU incorporation into tumors was determined (n = 5; *: p < 0.05).

(E) Ikkβ^f/f and Ikkβ^Δ dih12 cells were labeled with dsRed and 4 × 10⁵ cells were seeded into MUP-uPA mouse livers. Relative amounts of dsRed DNA in transplanted MUP-uPA livers were determined by real-time PCR 3 weeks post-inoculation and normalized to actin DNA as a measurement of cell growth (n = 5; *: p < 0.05).

(F) Ikkβ^Δ dih10 cells were reconstituted with a control vector or WT IKKβ expression vector and were s.c. injected into 8 weeks old C57BL/6 mice. Allograft volume was measured weekly (n = 5; *: p < 0.01).

(G) Ikkβ^f/f and Ikkβ^Δ dih10 cells were s.c. injected as above. Starting from day 2, mice were treated daily with MLN120B or vehicle by oral gavage. Tumor volume was measured weekly (n = 5; p < 0.01, f/f/control vs. f/f/MLN by one-way ANOVA).

Please also see Figure S3.