1. Volume 128, Issue 7, Pages 2054-2065 (June 2005)
Interleukin-6 Contributes to Mcl-1 Up-regulation and TRAIL Resistance via an Akt- Signaling Pathway in Cholangiocarcinoma Cells 
Shogo Kobayashi, Nathan W. Werneburg, Steven F. Bronk, Scott H. Kaufmann, Gregory J. Gores 
Gastroenterology 
Volume 128, Issue 7, Pages (June 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
Expression of Akt and Mcl-1 is increased in PSC and CCA. Immunohistochemistry for Akt and Mcl-1 was performed on liver transplant donor biopsy (control, n = 25) and liver tissue from patients with end-stage hepatitis C liver cirrhosis (n = 7), PSC (n = 29), and CCA (n = 33). (A) Representative photomicrographs are shown (original magnification, 200×). (B) Percent of patients with positive immunoreactivity for Akt or Mcl-1 compared with total number of cases. P < .01, PSC and CCA versus control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
Akt activation and Mcl-1 expression are IL-6 dependent in cholangiocarcinoma cells. (A) Immunoblot analysis of alpha chain (gp80) and beta chain (gp130) of IL-6 receptor was performed on whole-cell lysates (30 μg protein) from different cholangiocarcinoma cell lines. (B) KMBC cells were incubated for the indicated time in the presence or absence (control) of 5 μg/mL monoclonal antihuman IL-6-neutralizing antibodies. Whole-cell lysates were prepared, and aliquots containing 15 μg of protein were analyzed by immunoblot for Akt, phospho-Akt, and Mcl-1. Equal protein loading was verified by blotting for actin. (C) Immunoblot analysis of Akt, Phospho-Akt, and Mcl-1 was performed on whole-cell lysates (15 μg protein) from different CCA cell lines. Equal protein loading was verified by blotting for actin.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
Inhibition of Akt alters Mcl-1 expression. KMCH, KMBC, and Mz-ChA-1 cells were incubated for 12 hours in the presence or absence (control) of increasing concentrations of the Akt inhibitor A Whole-cell lysates were prepared, and aliquots containing 15 μg protein were analyzed by immunoblot for (A) Akt, phospho-Akt, GSK-3 beta, phospho-GSK-3 alpha/beta, and Mcl-1 or (B) p42/44 MAPK and phospho-p42/44 MAPK. Equal protein loading was verified by blotting for actin.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
Dominant negative Akt down-regulates Mcl-1. Cells were transfected with 10 μg of pCMV6 empty vector or dominant negative Akt (DN-Akt) in pCMV6. Twenty-four hours after transfection, whole-cell lysates were prepared, and aliquots containing 15 μg of protein were analyzed for Mcl-1 and actin.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
Akt activation in PI3K-mediated cells. Cells were cultured for 12 hours in the absence (control) or presence of 100 μmol/L LY Whole-cell lysates were prepared, and aliquots containing 15 μg of protein were analyzed by immunoblot for phospho-Akt, Mcl-1, and actin.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 6
Akt inhibition blocks Mcl-1 transcription. (A) KMBC cells were cultured for 6 hours in the absence or presence of the Akt inhibitor A (1.0 μmol/L). Total cellular RNA was isolated, and real-time reverse transcriptional-PCR using SYBR green as the fluorophore was performed using the LightCycler. The result was expressed as a relative ratio of Mcl-1 copies/mL to copies/mL of the housekeeping gene (18S) from the same RNA (respective complementary DNA) sample and PCR run (n = 4). *P < .05 versus control cells. (B) KMBC cells were cotransfected with 20 ng thymidine kinase-Renilla-cytomegalovirus and 1 μg pGL-3 Basic, pGL HindIII/PvuII, pGL-3 HindIII/XhoI, or pGL-3 HindIII/SpeI. Beginning 24 hours after transfection, cells were incubated with 1 μmol/L A or diluent for 12 hours. After firefly and Renilla luciferase activities were quantitated, data were expressed as the ratio of firefly luciferase activity/Renilla luciferase activity. Error bars represent mean ± standard deviations from 3 individual experiments. *P < .05; **P = .13 versus control cells. (C) KMBC cells were treated with medium containing 1.0 μmol/L A or diluent in the absence or presence of 5 μmol/L actinomycin D. At each indicated time, total cellular RNA was isolated, and real-time reverse-transcription PCR was performed as described in A. The results were expressed as percentage changes of Mcl-1 copies/mL as compared with that of time 0 sample of control. P > .5, control versus Akt inhibitor.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

8. Figure 7
Akt inhibition decreases Mcl-1 protein stability. KMBC cells were treated with medium alone (control) or medium containing A (1.0 μmol/L) in the presence of cycloheximide (20 μg/mL). (A) At each time point, cells were lysed and subjected to immunoblot analysis for Mcl-1 and actin. (B) Densitometric analysis of immunoblot in A was performed. The ratio Mcl-1/actin was calculated, assigning a value of 100% to the time 0 sample. The data are expressed as mean ± standard deviations from 3 separate experiments. *P < .1; **P < .01 versus control cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

9. Figure 8
Inhibition of Akt sensitizes cholangiocarcinoma cells to TRAIL-mediated apoptosis. Cells were treated with (A) TRAIL (2 ng/mL) for 12 hours in the presence or absence of monoclonal antihuman IL-6-neutralizing antiserum (5 μg/mL) for 48 hours and (B) TRAIL (1 ng/mL) in the presence or absence of Akt inhibitor A (1.0 μmol/L) for 12 hours. Apoptosis was quantitated by morphological criteria by fluorescence microscopy after 4′, 6-diamidino-2-phenylindole dihydrochloride staining. The data are expressed as mean ± standard deviations from 3 separate experiments. P < .01, TRAIL + anti-IL-6 versus TRAIL (Figure 8A) and (B) TRAIL + Akt inhibitor versus TRAIL.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions