Involvement of c-Myc in growth inhibition of Hep 3B human hepatoma cells by a vitamin K analog  Lisheng Ge, Ziqiu Wang, Meifang Wang, Siddhartha Kar, Brian I. Carr  Journal of Hepatology  Volume 41, Issue 5, Pages 823-829 (November 2004) DOI: 10.1016/j.jhep.2004.07.021 Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Cpd 5 decreases c-Myc protein levels. (A) Cpd 5 inhibits c-Myc protein time-dependently. Hep 3B cells were treated with Cpd 5 (15μM) from 15 to 240min. Whole cell lysates were resolved by SDS-PAGE and Western blot was performed using anti-c-Myc antibody. The blot was stripped and reprobed with β-actin antibody to confirm equal loading. (B) Cpd 5 inhibits c-Myc protein dose-dependently. Hep 3B cells were treated with Cpd 5 at different concentrations for 1h and cell lysates were then subjected to Western blot analysis using anti-c-Myc antibody. The blot was stripped and reprobed with β-actin antibody to confirm equal loading. Journal of Hepatology 2004 41, 823-829DOI: (10.1016/j.jhep.2004.07.021) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Cpd 5 induces c-Myc phosphorylation. (A) Cpd 5 induces c-Myc phosphorylation time-dependently. Hep 3B cells were treated with Cpd 5 (15μM) from 15 to 240min and cell lysates were then resolved by SDS-PAGE. Western blots were performed using anti-phospho-c-Myc antibody or anti-phospho-ERK antibody. The blot was stripped and reprobed with ERK2 antibody to confirm equal loading. (B) Cpd 5 induces c-Myc phosphorylation dose-dependently. Hep 3B cells were treated with different concentrations of Cpd 5, and Western blot analysis were performed using anti-phospho-c-Myc antibody or anti-phospho-ERK. The blot was stripped and reprobed with ERK2 to confirm equal loading. (C) MEK inhibitor antagonizes Cpd 5 effects. Hep 3B cells were treated with MEK inhibitor U0126 (10μM), or JNK inhibitor (20μM), for 1h, and then treated with Cpd 5 (15μM) for 1h. Western blots were performed on cell lysates using anti-phospho-c-Myc antibody. The same blot was stripped and reprobed with anti-c-Myc antibody. Journal of Hepatology 2004 41, 823-829DOI: (10.1016/j.jhep.2004.07.021) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Cpd 5-induced phospho-ERK phosphorylates c-Myc in vitro. (A) Cpd 5-induced cellular phospho-ERK phosphorylates c-Myc protein in vitro. Hep 3B cells were treated with Cpd 5 (20μM) for the indicated times and whole cell lysates were immunoprecipitated with immobilized anti-phospho-ERK antibody. Immunoprecipitated phospho-ERK was then incubated with c-Myc fusion protein. After incubation, Western blot was performed using anti-phospho-c-Myc antibody. (B) MEK inhibitor U0126 antagonizes phospho-ERK phosphorylation of c-Myc in vitro. Hep 3B cells were treated with MEK inhibitor U0126 (10μM) for 1h prior to Cpd 5 (20μM) addition. The immunoprecipitation and Western blot were performed as described above. Journal of Hepatology 2004 41, 823-829DOI: (10.1016/j.jhep.2004.07.021) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Cpd 5 induces GSK-3 activity. (A) Cpd 5 increases GSK-3 protein expression. Hep 3B cells were treated with Cpd 5 (15μM) for the indicated times and cell lysates were analyzed by Western blot using anti-GSK-3α or GSK-3β antibodies. The blot was stripped and reprobed with β-actin antibody to confirm equal loading. (B) GSK-3 inhibitor SB216763 antagonizes Cpd 5 action on c-Myc phosphorylation. Hep 3B cells were treated with MEK Inhibitor U0126 at 10μM, or GSK-3 inhibitor SB216763 at 5μM, or U0126 plus SB216763, for 1h, then Cpd 5 was added to the cell cultures at the final concentration of 15μM. Cell lysates were subjected to Western blot analysis using anti-c-Myc antibody. (C) GSK-3 kinase activity in vitro. Cell lysates from Cpd 5-treated Hep 3B cells were immunoprecipitated with anti-GSK-3α antibody, and immunoprecipitated GSK-3α was then incubated with c-Myc fusion protein as a substrate. The reaction was analyzed by Western blot using anti-phospho-c-Myc antibody. Journal of Hepatology 2004 41, 823-829DOI: (10.1016/j.jhep.2004.07.021) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 5 Cpd 5-induced GSK-3 activity enhances c-Myc protein degradation. (A) Proteasome inhibitor MG132 antagonizes Cpd 5 effects on c-Myc. Hep 3B cells were treated with MG132 at the final concentration of 40μM for 1h, and then Cpd 5 (15μM) was added to the cell cultures for 1h. Western blots were performed using anti-c-Myc antibody. The same blot was stripped and reprobed with anti-β-actin antibody to confirm equal loading. (B) GSK-3 inhibitor SB216763 antagonizes Cpd 5 effects on c-Myc levels. Hep 3B cells were treated with GSK-3 inhibitor SB216763 at 5μM for 1h, and then Cpd 5 (15μM) was added to the cell cultures for the indicated times. Western blots were performed using anti-c-Myc antibody (top row). The same blot was stripped and re-probed with anti-phospho-c-Myc antibody (middle row). The same blot was stripped again and probed with β-actin antibody to confirm equal loading (bottom row). Journal of Hepatology 2004 41, 823-829DOI: (10.1016/j.jhep.2004.07.021) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 6 Cpd 5 inhibits ODC protein levels and increases Gadd45 protein levels. (A) Cpd 5 reduces ODC protein levels. Hep 3B cells were treated with Cpd 5 (15μM) for the indicated times and cell lysates were subjected to Western blot using anti-ODC antibody. (B) Cpd 5 inhibits c-Myc DNA binding activity. Nuclear proteins were prepared from Hep 3B cells treated with Cpd 5 (15μM) for the indicated times, and c-Myc DNA binding was analyzed as described in the instruction manual of TransFactor Profiling Kit-Oncogenesis 2 from BD Biosciences Clontech. (C) Cpd 5 increases Gadd45 protein expression time-dependently. Hep 3B cells were treated with Cpd 5 (15μM) for the indicated times, and cell lysates were analyzed by Western blot using anti-Gadd45 antibody. The blot was stripped and reprobed with β-actin antibody to confirm equal loading. (D) Cpd 5 increases Gadd45 protein expression dose-dependently. Hep 3B cells were treated with Cpd 5 at different concentrations. Western blots were performed as described above. [This figure appears in colour on the web.] Journal of Hepatology 2004 41, 823-829DOI: (10.1016/j.jhep.2004.07.021) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions