1. Volume 44, Issue 2, Pages 342-349 (February 2006)
Interferon-α enhances TRAIL-mediated apoptosis by up-regulating caspase-8 transcription in human hepatoma cells 
Christian Liedtke, Nadine Gröger, Michael P. Manns, Christian Trautwein 
Journal of Hepatology 
Volume 44, Issue 2, Pages (February 2006)
DOI: /j.jhep
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

2. Fig. 1
Transcription of the caspase-8 gene is enhanced by IFNα via up-regulation of the caspase-8 promoter in Huh7 hepatoma cells. (A) Organisation of the human caspase-8 promoter. Predicted binding sites for STAT1/ISRE are indicated by an asteriks. (B). 24h after transfection with reporter plasmid −470/+76, Huh7 cells were stimulated with IFNα for the time indicated. Luciferase activity of cell extracts is depicted as fold induction compared to an un-stimulated control. The pGL2 luciferase vector was used as negative control. (C) Semi-quantitative duplex RT-PCR analysis of IFNα-treated Huh7 cells. Total RNA was subjected to duplex RT-PCR specific for caspase-8 and GAPDH. (D) Calculation of the caspase-8/GAPDH ratio.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

3. Fig. 2
Transcriptional up-regulation of caspase-8 by IFNα elevates procaspase-8 protein expression and is preceded by phosphorylation and nuclear transition of STAT1. (A) Caspase-8 protein expression following IFNα stimulation in Huh7 cells. Upper panel: Western blot for caspase-8 (55 and 54kD isoforms). Lower panel: expression of GAPDH (loading control). (B) STAT1 protein expression and phosphorylation in Huh7 cells after IFNα-treatment. Upper panel: STAT1 phosphorylated at Tyr701 (p-STAT1, α and β isoform). Medium panel: STAT1 expression in whole cell extracts. Lower panel: loading control (α-tubulin). (C) Construct −470/+76 was transfected into Huh7 cells. Where indicated, 1μg of a STAT1-expressing vector was co-transfected followed by IFNα stimulation for 8h.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

4. Fig. 3
Analysis of the caspase-8 promoter for functional IFNα responsive elements. (A) Upper panel: relevant promoter elements. Arrow: transcriptional start site. Lower panel: promoter fragments used for luciferase reporter studies. (B) Reporter plasmids depicted in (A) were transfected into Huh7 cells. Twenty-four hour after transfection cells were stimulated with IFNα for 8h. Luciferase activity is calculated as fold induction in comparison to an un-induced control.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

5. Fig. 4
Identification of a promoter element mediating the effect of IFNα on caspase-8 regulation. (A) Oligonucleotides used for EMSA. ISREpredicted: sequence −13 to +15 of the caspase-8 promoter; ISREmutated: introduction of a triple point mutation (underlined) in the ISRE site. (B) EMSA with IFNα-stimulated Huh7 cells using ISREpredicted. FP: free probe; time dependent formation of a new complex is indicated by an arrow. (C) Binding affinities of ISREpredicted (wt) and ISREmutated (mt) were compared in EMSA studies using nuclear extracts derived from Huh7 cells treated with IFNα for 24h. (D) ISRE mutation was introduced into plasmids −470/+76 and −52/+76. The resulting reporter plasmids −470/+76ISREmut and −52/+76ISREmut were transfected into Huh7 cells and stimulated with IFNα for 8h. Luciferase activity is shown as fold induction.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

6. Fig. 5
Co-operative effect of IFNα and TRAIL on apoptosis is mediated via a caspase-8 and caspase-3 dependent mechanisms. Huh7 hepatoma cells were treated with IFNα and TRAIL as indicated. (A) Determination of apoptosis. As controls untreated cells were investigated at the beginning (0) or 30h after onset of the experiment (30). (B) Determination of caspase-3 activity. (C) Huh7 cells were stimulated with TRAIL or TRAIL/IFNα. At time points indicated caspase-8 activity was determined. For each time point the caspase activity of TRAIL-stimulated cells was set to 100% and directly compared to the activity of cells co-stimulated with TRAIL and IFNα. (D) Determination of caspase-3 activity.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

7. Fig. 6
The synergistic effect of IFNα and TRAIL depends on enhanced caspase-8 expression and mitochondria permeability transition. (A) and (B) Huh7 cells were pre-treated with IFNα for 14h followed by stimulation with TRAIL for 3h. (A) Measurement of DNA fragmentation. (B) Determination of caspase-8 activity (fluorescence units). (C–F) Huh7 cells were transfected with 60 or 4pmol siRNA specific for caspase-8 (c8) or with 60pmol of a scrambled (scr) siRNA. 36h after transfection the cells were pre-treated with IFNα for 14h where indicated followed by a stimulation with TRAIL for 3h. (C) Determination of DNA fragmentation. (D) Measurement of caspase-8 activity (fluorescence units). Hatched bars: cells treated with scr siRNA. (E) Semi-quantitative duplex RT-PCR for caspase-8 and GAPDH (loading control). (F) Western blot analysis for caspase-8 protein expression and activation. The 55 and 54kD signals specific for pro-caspase-8 and the 28kD signal for the apoptosis-specific heterodimer are highlighted by arrows. (G) Huh7 cells were infected with recombinant adenoviruses expressing β-Galactosidase (adv-βGal) or Bcl2 (adv-Bcl2) and subsequently treated with IFNα and TRAIL as in (A) and (B). Upper panel: determination of DNA fragmentation. Black bars: β-Gal expressing control; grey bars: Bcl2-expressing cells. Lower panel: evidence of adenovirus-mediated Bcl2 expression.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions