Journal: American Journal of Physiology - Cell Physiology

Article Title: Induced ATF-2 represses CDK4 transcription through dimerization with JunD inhibiting intestinal epithelial cell growth after polyamine depletion

doi: 10.1152/ajpcell.00021.2010

Figure Lengend Snippet: Changes in the levels of total activating transcription factor-2 (ATF-2) and JunD proteins and their interactions after polyamine depletion in intestinal epithelial cell (IEC)-6 cells. Cells were grown in control cultures, cultures in which ornithin decarboxylase (ODC) activity inhibited by 5 mM α-difluoromethylornithine (DFMO), and cultures inhibited with DFMO and supplemented with 10 μM putrescine (Put) for 6 days. A: representative immunoblots of ATF-2 and JunD proteins. Whole cell lysates were harvested, applied to each lane (20 μg) equally, and subjected to electrophoresis on a 10% acrylamide gel. Levels of ATF-2 (∼70 kDa) and JunD (∼40 kDa) proteins were identified by a specific antibody (Ab) that recognizes ATF-2 or JunD. After the blots were stripped, actin (∼42 kDa) immunoblotting was performed as an internal control for equal loading. B: representative immunoblots of ATF-2 and JunD proteins in complexes immunoprecipitated (IP) by either anti-JunD (a) or anti-ATF-2 (b) antibody (Ab). After whole cell lysates (300 μg) were incubated with a specific Ab against JunD or ATF-2, precipitates were separated by performing electrophoresis on 10% acrylamide gels. Levels of ATF-2 and JunD were examined by Western immunoblotting with a specific anti-ATF-2 or anti-JunD Ab. Three experiments were performed that showed similar results.

Article Snippet: The antibody recognizing ATF-2, JunD, and CDK4 were from Santa Cruz Biotechnology (Santa Cruz, CA). α-Difluoromethylornithine (DFMO) was from Genzyme (Cambridge, MA).

Techniques: Activity Assay, Western Blot, Electrophoresis, Acrylamide Gel Assay, Immunoprecipitation, Incubation

Journal: American Journal of Physiology - Cell Physiology

Article Title: Induced ATF-2 represses CDK4 transcription through dimerization with JunD inhibiting intestinal epithelial cell growth after polyamine depletion

doi: 10.1152/ajpcell.00021.2010

Figure Lengend Snippet: Changes in cyclin-dependent kinase-4 (CDK4) gene expression in polyamine-deficient cells after ATF-2 silencing. A: effect of polyamine depletion on CDK4 transcription. a, levels of CDK4-promoter activity. Cells were grown in DMEM containing difluoromethylornithine (DFMO) alone or DFMO plus Put for 4 days and then transfected with the CDK4-promoter luciferase reporter construct. Luciferase activity was measured 48 h after the transfection in the presence or absence of DFMO or DFMO plus Put; data were normalized by Renilla-driven luciferase activity and expressed as means ± SE of data from 3 separate experiments. *P < 0.05 compared with control cells and cells exposed to DFMO plus Put. b, changes in mRNA levels of CDK4 in cells that were processed as described in Aa. Total cellular RNA was isolated, and levels of CDK4 mRNA were measured by using real-time quantitative PCR analysis. Values were means ± SE of data from 3 separate experiments. *P < 0.05 compared with control cells and DFMO plus Put cells. B: changes of CDK4 gene expression after ATF-2 silence in polyamine-deficient cells. a, representative immunoblots of ATF-2, JunD, and CDK4 proteins. After DFMO treatment for 4 days, cells were transfected with specific small interfering RNA (siRNA) targeting the coding region of ATF-2 mRNA (siATF-2) or control siRNA (C-siRNA), and whole cell lysates were harvested 48 h thereafter. The levels of ATF-2, JunD, and CDK4 were measured by Western blot analysis, and equal loading was monitored by actin immunoblotting. b, CDK4-promoter activity in cells described in Ba. Values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with control cells; +P < 0.05 compared with DFMO-treated cells transfected with C-siRNA. c, changes in levels of CDK4 mRNA in cells described in Ba. *P < 0.05 compared with controls, +P < 0.05 compared with DFMO-treated cells transfected with C-siRNA.

Article Snippet: The antibody recognizing ATF-2, JunD, and CDK4 were from Santa Cruz Biotechnology (Santa Cruz, CA). α-Difluoromethylornithine (DFMO) was from Genzyme (Cambridge, MA).

Techniques: Expressing, Activity Assay, Transfection, Luciferase, Construct, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Small Interfering RNA