Antibodies and Reagents
Anti-NPM monoclonal antibody (mAb) was kindly provided by Dr. P. K. Chan (Balyor College of Medicine, Houston, TX). Mouse monoclonal antibodies against E2F1, β-actin, retinoblastoma tumor suppressor protein (pRB), and rabbit polyclonal antisera against PP1α and PP1γ were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PP1β/δ rabbit polyclonal antibodies were purchased from Millipore (Billerica, MA). Rabbit antisera against phospho-NPM (Thr199), phospho-NPM (Thr234/237), and phospho-PP1α (Thr320) were from Cell Signaling Technology (Danvers, MA). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO), except where otherwise indicated.
Cell Culture and Transfection
HeLa, 293T, and H1299 cells were grown as a monolayer in DMEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin and streptomycin, in a 5% CO2-humidified incubator at 37°C. Cells were transfected using Lipofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Transient transfection was done for 24–54 h before cell harvest, unless otherwise noted.
Reporter Gene Assay
For the E2F1 (Lin et al., 2006
) and p53 (Chan et al., 2005
) promoter reporter assay, the cells extracts were obtained with 1× Reporter lysis buffer (Promega, Madison, WI), and the reporter/luciferase activity was measured using Luciferase assay reagent (Promega) following the manufacturer's recommendations. Luciferase activity was measured with Luminometer AutoLunmat LB953 (Berthold, Norwalk, CT) and normalized to the corresponding β-galactosidase activity.
UV and Phosphatase Inhibitor Treatments
Cells were grown to 80% confluence in 6-cm dishes. DNA damage was achieved by exposing the cells to UV irradiation (50 J/m2). Cells were harvested at indicated times. In some experiments, they were treated with okadaic acid (OA; 50 nM) or calyculin A (5 nM) for 3 h before UV treatment.
Western Blot Analysis
Cells were harvested and washed twice in phosphate-buffered saline (PBS) and then lysed in ice-cold Gold lysis buffer (1% Triton X-100, 10% glycerol, 1 mM sodium orthovanadate, 1 mM EGTA, 10 mM NaF, 1 mM sodium pyrophosphate, 10 μM β-glycerophosphate, 20 mM Tris-HCl, 137 mM NaCl, 5 mM EDTA, and cocktail protease inhibitor (Sigma-Aldrich), pH 7.9) for 30 min. Lysates were boiled in 2× urea sample buffer dye (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromphenol blue, 20% glycerol, 200 mM β-mercaptoethanol, and 8 M urea), and then fractionated by SDS-polyacrylamide gel electrophoresis (PAGE).
Western blot analysis was performed after electrophoretic separation of polypeptides by 8 or 10% SDS-PAGE and transfer to Hybond-polyvinylidene difluoride membrane (GE Healthcare, Piscataway, NJ). Blots were probed with the indicated primary and appropriate secondary antibodies. Immunobands were subsequently detected by the enhanced chemiluminescence reaction (GE Healthcare).
Generation of Small Interfering RNAs (siRNAs)
To establish a plasmid-based double-stranded RNAi system targeting endogenous PP1α, PP1β, or PP1γ, annealed oligonucleotides corresponding to partial sequence were designed and ligated to pSuper.neo+GFP (Oligoengine, Seattle, WA) according to manufacturer's instruction. The cDNA sequence of the targeted mRNA region for different genes is as follows: PP1α, 5′-GAGACGCTACAACATCAAA-3′; PP1β, 5′-GTTCGAGGCTTATGTATCA-3′; PP1β-2, 5′-TGTGCAGATGACTGAAGCA-3′, PP1γ, 5′-AGAGGCAGTTGGTCACTCT-3′; E2F1, 5′-CGCTATGAGACCTCACTGA-3′; and E2F1-2, 5′-GTGGACTCTTCGGAGAACT-3′.
To generate phosphorylation-site mutant constructs of NPM for expression in the cells, an appropriate set of oligonucleotide primers were used for site-directed amino acid substitutions (serine or threonine to alanine). We used full-length NPM cDNA in plasmid pCR3.1-FLAG-NPM as a template, and the primer sequences for cloning S125A, T199A, T199D, T234/237A, and T234/237D variants were listed in Supplemental Table S1.
The cells were harvested, washed twice in ice-cold PBS, and the cell pellets were resuspended in hypotonic RSB buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 1 mM phenylmethylsulfonyl fluoride) at 4°C for 10 min. An equal volume of an RSB + NP40 (20%) buffer was added next and incubated at 4°C for 10 min. Nuclei were collected by centrifuged at 3000 rpm at 4°C for 5 min and resuspended in ice-cold Gold lysis buffer (20 mM Tris-HCl, pH 7.5, 10% glycerol, 1% Triton X-100, 1 mM sodium pyrophosphate, 100 mM β-glycorophosphate, 1 mM sodium orthovanadate, 137 mM sodium chloride, 5 mM EDTA, 1 mM EGTA, and protease inhibitor cocktail) for 30 min and subsequently centrifuged at 12,000 rpm at 4°C for 30 min. Equal amounts of nuclei extract protein (1 mg) were incubated with the indicated antibodies (2 μg) at 4°C for 2 h with rotation. The immunocomplexes were captured with protein G-Sepharose (30 μl) for 2 h at 4°C with rotation. The protein G–antigen–antibody complexes were washed four times with the Gold lysis buffer and boiled in 2× urea sample buffer dye (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromphenol blue, 20% glycerol, 200 mM β-mercaptoethanol, and 8 M urea) for subsequent PAGE and immunoblotting analysis as described above.
Chromatin Immunoprecipitation (ChIP)
ChIP experiments were done essentially as described previously (Liu et al., 2007a
). Cross-linked, sonicated chromatin was precleared before being incubated with 2 μg of mouse mAb (pRB or E2F1) and rotated at 4°C overnight. Normal mouse immunoglobulin (Ig)G was used for the mock immunoprecipitation. After extensive washes, immunocomplexes were treated with proteinase K and decross-linked. Bound DNA in the pRB ChIP was extracted, purified, and subjected to polymerase chain reaction (PCR) analysis by using primers corresponding to the E2F1
gene promoter sequence (Lin et al., 2006
). After 35 cycles of amplification, PCR products were run on a 2% agarose gel and analyzed by ethidium bromide staining. For anti-E2F1 ChIP, bound DNA was subjected to quantitative real-time PCR analysis.
RNA Isolation and Reverse Transcription (RT)-PCR
Total cellular RNAs were isolated from cells using the Trizol Reagent (Invitrogen) according to the manufacturer's instructions. Total RNA (2–5 μg) was reverse transcribed with reverse transcriptase (Moloney murine leukemia virus) (Invitrogen) at 37°C for 52 min. cDNA synthesis was primed with random hexamer. The E2F1 and internal control β-actin genes were amplified with the following specific primers: E2F1, 5′-CCGAGGTGCTGAAGGTGCAGA-3′ (sense), 5′-TCTTCCCAGGGCTGATCCCAC-3′ (antisense); and β-actin, 5′-AGAAAATCTGGCACCACACC-3′ (sense), 5′-CCATCTCTTGCTCGAAGTCC-3′ (antisense). The PCR products were resolved on 1.5% agarose gels and stained with ethidium bromide.
For quantitative determination by real-time PCR, target transcripts or ChIP products were analyzed using the LightCycler system and Power SYBR Green PCR Master Mix (Roche Diagnostics, Indianapolis, IN). Transcript levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) values of the respective sample, and represent mean ± SD of at least three independent experiments. Sequences for the primers used in this experiment are as follows: E2F1, 5′-AGATGGTTATGGTGATCAAAGCC-3′ (sense), 5′-ATCTGAAAGTTCTCCGAAGAGTCC-3′ (antisense); GAPDH, 5′-GGTATCGTGGAAGGACTCATGAC-3′ (sense), 5′-ATGCCAGTGAGCTTCCCGT-3′ (antisense); XPC, 5′-CATCATCCCAGCCCGCTTTAC-3′ (sense), 5′-CCACTTCACCAGGTTTGAGAGG-3′ (antisense); DDB2, 5′-GCACCTCACACCTATCAAG-3′ (sense), 5′-GAGCTGACACATCATCTTCC-3′ (antisense); and RPA3, 5′-CGTCACTAAGCAGCCAATC-3′ (sense), 5′-GCACCAATCAGCGAAGAC-3′ (antisense). For quantitative analysis of anti-E2F1 ChIP experiments, primers corresponding to gene promoters were used: XPC, 5′-GAAATAGAGAGAAACCTGTTGT-3′ (sense), 5′-CTAGTCACGCCCCTAAAG-3′ (antisense); and DDB2, 5′-ATGTTTGGCGGGAAGTTG-3′ (sense), 5′-TCTGGGGAGAAACAAGGC-3′ (antisense). The results are given as a percentage of input and represent mean ± SD of at least three independent experiments. Triplicate PCRs were performed for all experiments.
In Vitro Phosphatase Assay
The cells were okadaic acid treated (50 nM) for 8 h before harvest. Nuclei extracts were prepared and subjected to immunoprecipitation by using the anti-NPM antibody. The immunocomplexes were washed with reaction buffer (1 mM MnCl2 and 5 mM caffeine). One unit of PP1 (New England Biolabs, Ipswich, MA) was added to the immunocomplexes for 1 h at 30°C. For inhibition studies, the immunocomplexes and PP1 were incubated in the presence of 1 μM OA. Degree of dephosphorylation was analyzed by SDS-PAGE and subsequently immunoblotting.
Measurement of Thymine Dimers
To estimate the DNA repair capacity of cells, the amounts of thymine dimers in UV-irradiated cells were measured by enzyme-linked immunosorbent assays (ELISAs) as described previously (Zhai et al., 2005
) using an anti-thymine dimer antibody (Abcam, Cambridge, MA). DNA was extracted from the cells at the indicated times postirradiation. For statistical significance of quantitative comparisons, calculations were done by the SigmaPlot software (Systat Software, San Jose, CA).
DNA Repair Capacity/Host Cell Reactivation (HCR) Assay
This method for measuring nucleotide excision repair (NER) in response to UV irradiation was performed based on a previous report (Athas et al., 1991
). The pCAT control vector (Promega) is a nonreplicated plasmid, under control of simian virus 40 promoter and enhancer sequences. The plasmid was treated with or without UV (254 nm) at the dose of 500 J/m2
and then cotransfected with unirradiated pGL3luc (as an internal control) as well as plasmids encoding various phosphorylation-site mutants of NPM into HeLa cells. The cells were lysed in 1× Reporter lysis buffer 24 h after transfection, and chloramphenicol acetyltransferase (CAT) activity was detected as described previously (Wu et al., 2002a
). After normalization, relative CAT activity was expressed as the percent of activity in cells transfected with the UV-irradiated plasmid over those harboring the undamaged plasmid. For statistical significance of quantitative comparisons, calculations were done by the SigmaPlot software.