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Unbound E2F modulates TGF-ß1-induced apoptosis in HuH-7 cells

¹ Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455, USA
² Department of Genetics, Cell Biology, and Development, University of Minnesota Medical School, Minneapolis, MN 55455, USA

View larger version (45K): [in a new window]   Fig. 1. Morphological and cell cycle analyses of synchronized G1 and TGF-ß1-induced apoptotic HuH-7 cells. The cells were treated with 1 nM TGF-ß1 for 72 hours (left panels) or synchronized at G1 (right panels) as described in Materials and Methods. (A) Fluorescent labeling of nuclei with Hoechst 33258 showing the characteristic nuclear fragmentation associated with TGF-ß1-induced apoptosis (left). (B) Analysis of cell cycle phase by flow cytometry after removing apoptotic cells. The percentage of total cells present in the different cell cycle phases is indicated in the top-right panel.

View larger version (100K): [in a new window]   Fig. 2. E2F mobility-shift assays of nuclear extracts from synchronized G1 and TGF-ß1-induced preapoptotic cells. Nuclear extracts were prepared, incubated with wild-type 32P-labeled E2F oligonucleotide probe and analyzed by PAGE (lanes 1-12) as described in Materials and Methods. Unlabeled wild-type E2F oligonucleotides were added at 10-fold (lanes 5,9), 100-fold (lanes 6,10) and 1000-fold (lanes 7,11) molar excess, or a 1000-fold molar excess of unlabeled mutant E2F oligonucleotide (lanes 8,12) during the 30 minute incubation for binding specificity. Negative controls containing no nuclear extracts were devoid of activity (lanes 13,14). The position and numerical designation of the 32P-labeled DNA—protein complexes are indicated on the left. The cell-cycle-associated phases of the nuclear extracts are indicated at the top. mt, mutant E2F oligonucleotide; TGF-ß1, preapoptotic nuclear extract from HuH-7 cells incubated with TGF-ß1 for 72 hours; wt, wild-type E2F oligonucleotide.

View larger version (32K): [in a new window]   Fig. 3. E2F-1 expression in synchronized or TGF-ß1-treated preapoptotic G1 cells determined by northern or western blot analysis. (A) Representative northern blot of 15 µg of total RNA hybridized with a 0.7 kb EcoRI/BglII E2F-1 cDNA fragment labeled with digoxigenin-dUTP detected using the DIG/GeniusTM system as described in Materials and Methods. (B) Whole cell lysates were prepared and subjected to western blot analysis using primary monoclonal C-20 anti-E2F-1 antibody. The proteins were detected using a horseradish peroxidase conjugated secondary antibody and ECLTM as described in Materials and Methods. The cell cycle phase is indicated at the top. TGF-ß1, preapoptotic cells following 72 hours of incubation with TGF-ß1.

View larger version (96K): [in a new window]   Fig. 4. Dissociation of protein-protein interactions in E2F—DNA—protein complexes. Nuclear extracts were prepared from synchronized G1 and TGF-ß1-induced preapoptotic cells. DNA-binding assays were performed using 32P-labeled wild-type E2F-specific oligonucleotides with increasing concentrations of sodium deoxycholate from 0.2% to 1.2%. The DNA—protein complexes were analyzed by PAGE as described in Materials and Methods. The position and numerical designation of the 32P-labeled DNA—protein complexes are indicated on the left. The cell cycle phase from which the nuclear extract was obtained is shown at the top. DOC, sodium deoxycholate; TGF-ß1, preapoptotic nuclear extracts from HuH-7 cells incubated for 72 hours with 1 nM TGF-ß1.

View larger version (65K): [in a new window]   Fig. 5. Determination of proteins bound to E2F derived from nuclear extracts of G1 and preapoptotic cells. The nuclear extracts were prepared from synchronized G1 and TGF-ß1-induced preapoptotic cells. Aliquots were mixed with a 32P-labeled E2F-specific oligonucleotide probe and antibodies to either pRb, E2F-1, -2, or -3 (A), as well as known E2F-binding proteins, DP-1 and -2, the pRb family members p107 and p130 and the oncogene MDM2 (B). The super-shifted DNA—protein complexes were analyzed by PAGE as described in Materials and Methods. The position and numerical designation of the 32P-labeled DNA—protein complexes are indicated on the left and right. The associated cell cycle phases of the nuclear extracts are shown at the top as well as the specific antibody used for the super-shift analysis. The numerical designations for the lanes referred to in the text are indicated at the bottom. G1, nuclear extract from G1 synchronized normal cells; Tß, nuclear extract from TGF-ß1 preapoptotic G1 cells.

View larger version (18K): [in a new window]   Fig. 6. Increased E2F activity is associated with elevated levels of E2F-mediated transcription. (A) The CAT expression plasmids E2F1CAT and 4XE2FCAT under E2F-dependent promoters as well as ß-galactosidase control constructs were co-transfected into HuH-7 cells as described in Materials and Methods. 24 hours post-transfection, the cells were cultured in the presence (+) or absence (-) of 1 nM TGF-ß1 for an additional 36 hours before harvesting the cells for determination of CAT and ß-galactosidase activity. (B) The E2F-controlled CAT constructs were co-transfected into the cells with (+) or without (-) pCMV.RB expression plasmids. After 24 hours, TGF-ß1 was added to the incubation media and the cells harvested for CAT analysis as indicated above. The results, normalized to ß-galactosidase expression, are the mean determinations of at least three different experiments, each done in duplicate.

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