Figure 1.
IFNs down-regulate β-catenin signaling pathway.
HCC cell lines, HepG2 (A) and Huh7 (C), were left untreated or treated with IFNα (100 ng/ml), IFNγ (100 ng/ml) or IFNλ (100 ng/ml) for 24 hrs prior to transfection with TOPflash luciferase and Renilla luciferase constructs. After resting for 4 hrs, the cells were cultured with or without initial treatment of different IFNs. Dual luciferase activity was measured 24 hrs later. Data shown is normalized to Renilla activity. HepG2 (B) and Huh7 (D) were treated with or without IFNα (100 ng/ml), IFNγ (100 ng/ml) or IFNλ (100 ng/ml) for 48 hrs and expression of hypophosphorylated/active β-catenin level was measured by conventional intracellular flow cytometry. Data represent a minimum of three experiments and asterisks denote p<0.05 in comparison to untreated samples.
Figure 2.
Impact of IFN-mediated β-catenin regulation on proliferation and apoptosis in HCC.
HepG2 (A) and Huh7 (D) cells were left untreated or treated with IFNα (100 ng/ml), IFNγ (100 ng/ml) or IFNλ (100 ng/ml) for 72 hrs. Cell viability was determined at 72 hrs. The abscissa represents the types of stimulation. The ordinate represents percentage of live cells relative to the mock treated cells. Apoptosis in HepG2 (B, C) and Huh7 (E, F) was measured by TUNEL assay and flow cytometry targeting active caspase 3. The ordinate represents fold increase of fluorescence intensity relative to the untreated cells. Data represent a minimum of three experiments and asterisks denote p<0.05 in comparison to untreated samples.
Figure 3.
IFNs induce DKK1 expression and have no effects on GSK3β.
HepG2 and Huh7 cells were treated with or without IFNα (100 ng/ml), IFNγ (100 ng/ml) or IFNλ (100 ng/ml) and expression of DKK1 was measured by flow cytometry (A, C) or ELISA (B, D) at 48 hrs post-treatment. Active GSK3β level was measured in HepG2 and Huh7 cells at 48 hrs post different IFN exposure by flow cytometry (E, F). Data represent a minimum of three experiments and asterisks denote p<0.05 in comparison to untreated samples.
Figure 4.
IFNs induce activation of STAT1 and STAT3 in HCC.
HepG2 cells were left untreated or treated with IFNα, IFNγ or IFNλ for 0.5 hr in the absence (black bars) or presence of STAT1 inhibitor (FLUD, grey bars) or STAT3 inhibitor (S3I, open bars), and active STAT1 (A) and STAT3 (B) levels were measured by flow cytometry. Data represent a minimum of three independent experiments. Asterisks denote p<0.05 in comparison to untreated samples.
Figure 5.
IFN-induced up-regulation of DKK1 is dependent on STAT3 activation.
HepG2 (A) and Huh7 (B) cells were left untreated or treated with FLUD, S3I, or αDKK1 (DKK1 neutralizing antibody) alone or in combination with IFNα, IFNγ or IFNλ, respectively for 48 hrs. DKK1 levels were measured by ELISA. Data represent a minimum of three independent experiments. Asterisks denote p<0.05 in comparison to untreated samples.
Figure 6.
IFN-induced apoptosis in HCC is DKK1 and STAT3-dependent.
HepG2 (A) and Huh7 (B) cells were left untreated or treated with FLUD, S3I, or αDKK1 alone or in combination with IFNα, IFNγ or IFNλ, respectively for 72 hrs. The levels of induced apoptosis were measured by TUNEL assay. Data represent a minimum of three independent experiments. Asterisks denote p<0.05 in comparison to untreated samples.