Cell lines, synchronization and drug treatment.
HeLa cervical carcinoma cells and T98G glioblastoma cells obtained from ATCC were cultured in Dulbecco's modified Eagle's medium (DMEM) plus 10% FBS at 37°C with 5% CO2. Cells were synchronized at G1/S using a double thymidine block. Briefly, HeLa cells were treated with 2 mM thymidine (Sigma) for 19 h, washed with PBS and cultured in fresh media for 9 h. The cells were treated again with thymidine for 16 h and were released from the block by washing in PBS and replacing the media with fresh DMEM + 10% FBS. Synchronization at prometaphase with nocodazole was performed by incubating cells for 16 h with 330 nM nocodazole (Sigma). The protein synthesis inhibitor cycloheximide (Sigma) was dissolved in H2O.
Mammalian expression plasmids and transfection.
HA-E2F1 and HA-DP1 have been described previously.22
The HA-tagged Cdh1, HA-tagged Cdc20, FLAG-tagged Cdh1, FLAG-tagged Cdc20 wild-type, FLAG-tagged Cdc20 ΔN165 and FLAG-tagged Skp2 plasmids were provided by M. Pagano (NYU School of Medicine, New York, NY). FLAG-tagged β-Trcp1 was provided by A. Abeliovich (Columbia University, New York, NY). pCMV-Rb plasmid was a kind gift of Dr. Bill Kaelin (Dana-Farber Cancer Institute, Boston, MA). E2F1 D-box mutants were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturers instructions. All constructs were confirmed by sequencing. Transient cell transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturers instructions.
Cell cycle analysis.
Cellular DNA content was assessed by propidium iodide (PI) staining as follows. Cells were harvested, washed twice with PBS, fixed in 50% ice-cold ethanol for 30 min on ice, washed twice again with PBS and resuspended in PBS containing 66 µg/mL PI (Sigma) and 100 µg/mL RNaseA (Sigma). Cells were analyzed by flow cytometry on a FACScan cytometer (BD Biosciences) using CellQuest software. The percentage of cells in each cell cycle phase was quantified using the ModFit program.
Western blot analysis and immunoprecipitations.
For immunoblotting, cells were washed once in PBS, resuspended in TEGN buffer (10 mM Tris at pH 7.5, 1 mM EDTA, 10% glycerol, 0.5% NP40, 400 mM NaCl, 1 mM DTT, 0.5 mM phenylmethylsulfonylfluoride and protease inhibitor mixture containing 1 M Benzamidine, 3 mg/mL Leupeptin, 100 mg/mL Bacitracin and 1 mg/mL α2-macroglobulin) and incubated on ice for 30 min. Lysates were then cleared by centrifugation at 13,000 rpm for 5 min. Total protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Laboratories), and equal amounts of protein were separated on 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes (Protran, Schleicher and Schuell). Membranes were then blocked for 1 h in 5% skim milk in 0.5% tween-PBS (tPBS) and then probed with primary antibody overnight at 4°C in tPBS. After washing 3 × 7 min with tPBS, membranes were incubated for 1 h with secondary horseradish peroxidase-conjugated antibody (Sigma) in tPBS. Following 3 × 7 min washes in tPBS, proteins were visualized by enhanced chemiluminesence (Amersham Biosciences). For immunoprecipitations, cells were lysed in TEGN buffer and 300 µg of protein/sample was incubated with 0.5 µL anti-FLAG M2 antibody (Sigma) for 16 h at 4°C. 10 µl of a 50/50 protein A-Sepharose (GE Healthcare) slurry and 20 µl of a 50/50 protein G-Sepharose (GE Healthcare) slurry were added for an additional hour before washing extensively in TEGN buffer. Bound protein was eluted by boiling in SDS sample buffer and immunoprecipitates were analyzed by immunoblotting as described above.
The following primary antibodies were used: anti-E2F1 (C20 or KH-95, 1:1,000, Santa Cruz Biotechnology), anti-HA (HA.11, 1:1,000, Covance), anti-Flag M2 (1:2,000, Sigma), anti-MYC (9E10, 1:500, Santa Cruz Biotechnology), anti-DP1 (TFD10, 1:1,000, BD Biosciences), anti-Rb (IF-8, 1:5, supernatant from hybridoma culture), anti-Cdc20 (H-175, 1:1,000, Santa Cruz Biotechnology), anti-Cdh1 (DH01, 1:1,000, NeoMarkers), anti-cyclin B (GNS1, 1:1,000, Santa Cruz Biotechnology) and anti-actin (1:5,000, Sigma).
siRNA duplexes were synthesized by Qiagen. The siRNA oligonucleotide sequences for Cdh1 were: 5′-(UGA GAA GUC UCC CAG UCA G)dTdT-3′ (sense) and 5′-(CUG ACU GGG AGA CUU CUC A)dTdT-3′ (anti-sense). The siRNA sequences for Cdc20 were: 5′-(CGG CAG GAC UCC GGG CCG A)dTdT-3′ (sense) and 5′-(UCG GCC CGG AGU CCU GCC G)dTdT (anti-sense). The siRNA sequences for Luc were: 5′-(CUU ACG CUG AGU ACU UCG A)dTdT (sense) and 5′-(UCG AAG UAC UCA GCG UAA G)dTdT (anti-sense). For siRNA transfection, cells were transfected with 50 nM siRNA using DharmaFECT 1 siRNA transfection reagent (Dharmacon) according to the manufacturers instructions.
In vivo ubiquitination experiments.
H1299 cells were co-transfected either with plasmids encoding Myc-E2F1, HA-ubiquitin or FLAG-Cdc20 (wild type or mutants). At 24 h post-transfection cells were harvested and Myc-E2F1 was immunoprecipitated using anti-Myc antibody. Immunoprecipitates were subjected to SDS-PAGE, transferred to nitrocellulose and both immuonoprecipitates and whole-cell lysates were blotted with anti-HA antibody for HA-tagged ubiquitin.
siRNA-transfected cells were harvested, washed with PBS, lysed in SB buffer (25 mM HEPES pH 7.5, 1.5 mM MgCl2
, 5 mM KCl, 1 mM dithiothreitol, 15 mM creatine phosphate, 2 mM ATP, 0.5 mM phenylmethylsulfonylfluoride and protease inhibitor mixture containing 1 M Benzamidine, 3 mg/mL Leupeptin, 100 mg/mL Bacitracin and 1 mg/mL α2
macroglobulin) and homogenized by freeze-thawing and passage through a needle. Extracts were then subjected to centrifugation (5 min at 5,000 rpm followed by 60 min at 13,000 rpm), and total protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Laboratories). Extracts (30 µg/sample) were supplemented with degradation cocktail [1.5 mg/mL ubiquitin (PK-ubiquitin, prepared as previously described39
), 7.5 mM creatine phosphate, 1 mM ATP, 1 mM MgCl2
and 0.1 mg/mL cycloheximide] and incubated at room temperature. Aliquots were collected at 0, 1, 2, 4 and 6 h for immunoblotting as described above.
RNA preparation and quantitative real-time PCR.
Total RNA was extracted from HeLa cells using the Qiagen RNeasy kit according to the manufacturer's instructions. RNA was additionally treated with RNase-free DNase I (Qiagen) to remove any residual genomic DNA. First-strand cDNA synthesis was performed with the Superscript III Supermix for qRT-PCR kit (Invitrogen) according to the manufacturer's instructions. Each PCR was carried out in triplicate in a 20 µL volume using Power SYBR Green PCR Master Mix (Applied Biosystems) for 15 min at 95°C for initial denaturing, followed by 35 cycles of 95°C for 30 s and 60°C for 30 s in the ABI Prism 7300 Real-Time PCR System. The ribosomal gene L32 was used as the control gene. Values for each gene were normalized to the expression levels obtained for L32, and fold induction was calculated versus time 0 h. Each reaction was done in triplicate from at least two independent experiments. Primer sequences are available upon request.