Journal: Journal of Biological Chemistry

Article Title: Ubiquitin/Proteasome-dependent Degradation of D-type Cyclins Is Linked to Tumor Necrosis Factor-induced Cell Cycle Arrest

doi: 10.1074/jbc.m109929200

Figure Lengend Snippet: FIG. 2. TNF inhibits the expression of D-type cyclins and their kinase activities. TF-1 and MV4–11 cells treated with or without TNF (30 ng/ml) were collected and lysed in lysis buffer. A, aliquots of lysates were analyzed for the expression of cyclins and cdks by Western blotting with the antibodies indicated in the figure. B, TF-1 cells treated with TNF for 48 h were collected and lysed in lysis buffer. Aliquots of lysates containing 200 g of total proteins were immunoprecipitated with antibodies against cyclins D2, D3, cdk4, or IgG. The immunopre- cipitates were then incubated with GST-Rb in kinase buffer in the presence of 10 Ci of [-32P]ATP and the expression of phosphorylated GST-Rb was detected by autoradiography. Expressed proteins were quantitated by image reading, using ImageQuant software, and percent increase or decrease was calculated as described under “Experimental Procedures.”

Article Snippet: Reagents—Antibodies used and their sources were as follows: antibodies to I B , cyclin-dependent kinase 2 (cdk2), cdk4, cdk6, cyclin A, cyclin E, cyclin D1, cyclin D2, cyclin D3, p27, and actin (Santa Cruz Biotechnology, Santa Cruz, CA); pRb, phosphorylated pRb, and luciferase (Promega, Madison, WI).

Techniques: Expressing, Lysis, Western Blot, Immunoprecipitation, Incubation, Autoradiography, Software

Journal: Journal of Biological Chemistry

Article Title: Ubiquitin/Proteasome-dependent Degradation of D-type Cyclins Is Linked to Tumor Necrosis Factor-induced Cell Cycle Arrest

doi: 10.1074/jbc.m109929200

Figure Lengend Snippet: FIG. 3. TNF-induced down-regulation of D-type cyclins is caspase-3 pathway independent. A, TF-1 cells were pretreated with or without zVAD-FMK (100 M), followed by addition of TNF (20 ng/ml) for 24 h. As a negative control, growing cells were treated with dimethyl sulfoxide (the vehicle for zVAD-FMK) alone at the same condition. Subsequently, cell lysates were prepared and aliquots of the lysates were analyzed by Western blotting with antibodies against cyclin D2, cyclin D3, caspase 3, or activated caspase 3. B, cells were treated with or without TNF in the presence or absence of zVAD-FMK for 24 h, after which cells were collected and stained with propidium iodide for cell cycle analysis by flow cytometry. C, cells in log phase treated with different concentrations of TNF for 24 h were collected and lysed, and an aliquot of lysate was analyzed for the expression of cyclin D3 by Western blotting with anti-cyclin D3 antibody. The relative inhibition was calculated from quantitation of individual bands after subtraction from control bands. In a parallel experiment, some cells after treatment with TNF were incubated with trypan blue for 15 min at room temperature. A drop of the cell suspension was loaded on a hemocytometer, and stained (blue) and unstained cells were counted under a light microscope. Cell viability was calculated as described under “Experimental Procedures.” Some cells treated with 10 ng/ml TNF were collected, lysed, and analyzed for the expression of cyclin D3 and activated caspase 3 with antibodies against cyclin D3 or caspase 3, respectively.

Article Snippet: Reagents—Antibodies used and their sources were as follows: antibodies to I B , cyclin-dependent kinase 2 (cdk2), cdk4, cdk6, cyclin A, cyclin E, cyclin D1, cyclin D2, cyclin D3, p27, and actin (Santa Cruz Biotechnology, Santa Cruz, CA); pRb, phosphorylated pRb, and luciferase (Promega, Madison, WI).

Techniques: Negative Control, Western Blot, Staining, Cell Cycle Assay, Flow Cytometry, Expressing, Inhibition, Quantitation Assay, Control, Incubation, Suspension, Light Microscopy

Journal: Journal of Biological Chemistry

Article Title: Ubiquitin/Proteasome-dependent Degradation of D-type Cyclins Is Linked to Tumor Necrosis Factor-induced Cell Cycle Arrest

doi: 10.1074/jbc.m109929200

Figure Lengend Snippet: FIG. 4. TNF accelerates proteolytic degradation of D-type cy- clins. A, total RNA (10 g/reaction) extracted from MV4–11 cells treated with or without TNF were hybridizated with RNA probe con- taining the specific mRNAs for the different cyclins as indicated in the figure. After hybridization and RNase treatment, the protected RNAs were separated by SDS-PAGE and detected by autoradiography. The housekeeping gene probes, L32 and GAPDH, are included among the RNA probes for normalizing sampling and technique errors and to permit comparison of individual mRNA species between samples. Iden- tical results were obtained from four separate experiments. B, MV4–11 cells were treated with or without 30 ng/ml TNF for 24–48 h. The cells were metabolically labeled with [35S]methionine and immunoprecipi- tated cyclins or cdk were resolved by SDS-PAGE and visualized by autoradiography. In parallel experiments, MV4–11 cells were treated with or without 30 ng/ml TNF for 48 h. The cells were then labeled with [35S]methionine for 1 h and subsequently chased by RPMI containing unlabeled methionine for various times as indicated in the figure. Immunoprecipitated cyclins were resolved by SDS-PAGE and visual- ized by autoradiography. C, the quantitation of individual bands under “degradation” in B was calculated as percent expression after subtrac- tion of background by image reading, using ImageQuant software.

Article Snippet: Reagents—Antibodies used and their sources were as follows: antibodies to I B , cyclin-dependent kinase 2 (cdk2), cdk4, cdk6, cyclin A, cyclin E, cyclin D1, cyclin D2, cyclin D3, p27, and actin (Santa Cruz Biotechnology, Santa Cruz, CA); pRb, phosphorylated pRb, and luciferase (Promega, Madison, WI).

Techniques: Hybridization, SDS Page, Autoradiography, Sampling, Comparison, Metabolic Labelling, Labeling, Immunoprecipitation, Quantitation Assay, Expressing, Software

Journal: Journal of Biological Chemistry

Article Title: Ubiquitin/Proteasome-dependent Degradation of D-type Cyclins Is Linked to Tumor Necrosis Factor-induced Cell Cycle Arrest

doi: 10.1074/jbc.m109929200

Figure Lengend Snippet: FIG. 5. TNF-induced down-regulation of D-type cyclins is via proteasome/proteolysis-dependent degradation. MV4–11 cells were pretreated without or with ALLN (1 g/ml), MG132 (1 M), lac- tacystin (2 M), or zVAD-FMA (100 M) for 30 min, followed by addition of TNF for 30 min (for detection of IB and NF-B) or 48 h (for detection of D-type cyclins), after which cells were collected and lysed in lysis buffer. Lysates (nuclear lysates for NF-B and whole lysates for IB and D-type cyclins) containing 50 g of total proteins were sepa- rated on 10–12% SDS-PAGE and analyzed for the expression of IB, NF-B (p65), cyclin D2, and cyclin D3 with an antibody against each of these molecules (A). In parallel experiments, lysates containing 200 g of total protein were immunoprecipitated with antibodies against cyc- lins D2 and D3, after which the immunoprecipitates were measured for their kinase activities by in vitro kinase assay, using GST-Rb as a substrate. The relative kinase activity is calculated from quantitation of individual bands after subtraction of background by image reading using ImageQuant software (B). 1 and 4, control cells; 2 and 5, cells treated with TNF (30 ng/ml) for 48 h; 3 and 6, cells pretreated with MG132 (1 M) followed by addition of TNF (30 ng/ml) for 48 h. The kinase activity from control cells was set as 100%. Similar results were obtained from three (A) or two (B, mean S.D.) separate experiments. Expressed proteins were quantitated by image reading, using Image- Quant software, and percent increase or decrease was calculated as described in the legend for Fig. 2.

Article Snippet: Reagents—Antibodies used and their sources were as follows: antibodies to I B , cyclin-dependent kinase 2 (cdk2), cdk4, cdk6, cyclin A, cyclin E, cyclin D1, cyclin D2, cyclin D3, p27, and actin (Santa Cruz Biotechnology, Santa Cruz, CA); pRb, phosphorylated pRb, and luciferase (Promega, Madison, WI).

Techniques: Lysis, SDS Page, Expressing, Immunoprecipitation, In Vitro, Kinase Assay, Activity Assay, Quantitation Assay, Software, Control

Journal: Journal of Biological Chemistry

Article Title: Ubiquitin/Proteasome-dependent Degradation of D-type Cyclins Is Linked to Tumor Necrosis Factor-induced Cell Cycle Arrest

doi: 10.1074/jbc.m109929200

Figure Lengend Snippet: FIG. 7. In vitro degradation of cyclin D3 by 26 S proteasome. Human cyclin D3 plasmid (pRc/CMV/cyclin D3) was transcribed and translated under control of T7 RNA polymerase promoter using TNT transcription/translation kit (Promega) following the manufacturer’s instruction. pRc/CMV alone and luciferase DNA were used as negative and positive controls, respectively. An aliquot of translation product (2 l) was loaded to SDS-PAGE and analyzed for the expression of cyclin D3 and luciferase by Western blotting with antibodies against cyclin D3 and luciferase, respectively (A). Subsequently, aliquots of the translation products were conjugated with ubiquitin in the conjugation buffer containing ATP, E1, E2, E3, and ubiquitin following the manufacturer’s protocol (Calbiochem). Aliquots of cyclin D3 proteins conjugated with or without ubiquitin were incubated with Mg/ATP and 26 S proteasome at 37 °C for 1 h before addition of quenching buffer and centrifugation following the manufacturer’s instruction (Calbiochem). Aliquots of degradation products were loaded to SDS-PAGE and analyzed for the expression of cyclin D3 with an antibody against cyclin D3 (B).

Article Snippet: Reagents—Antibodies used and their sources were as follows: antibodies to I B , cyclin-dependent kinase 2 (cdk2), cdk4, cdk6, cyclin A, cyclin E, cyclin D1, cyclin D2, cyclin D3, p27, and actin (Santa Cruz Biotechnology, Santa Cruz, CA); pRb, phosphorylated pRb, and luciferase (Promega, Madison, WI).

Techniques: In Vitro, Plasmid Preparation, Control, Luciferase, SDS Page, Expressing, Western Blot, Ubiquitin Proteomics, Conjugation Assay, Incubation, Centrifugation