Plasmids and antibodies

Yeast constructs expressing wild-type (WT) and SIM mutated (DM) Gal AD-FAF, mammalian vectors expressing EGFP-SUMO1, HA-FAF1, Flag-FAF1 WT and DM were described previously [24]. Mammalian expression vectors encoding HA-tagged human SAP130 and HDAC1 (HG17174-NY and HG11486-NY) were purchased from Sino Biological Inc. (Beijing, China). pCS2 + MT-mSin3A was a gift from Bob Eisenman (Addgene plasmid # 30,452; http://n2t.net/addgene:30452; RRID: Addgene_30452). Myc-tagged ubiquitin was cloned into the pCMV-Tga3C vector (Stratagene). A polymerase chain reaction (PCR) fragment encoding the C-terminal of human SAP130 from HA-SAP130 was subcloned into the pBTM116 vector in frame with the LexA domain to generate the LexA-SAP130 bait. SAP130 mutations at three potential SUMO-conjugated Lys residues were created in the pBTM116 and pCMV3 using the Quikchange site-directed mutagenesis kit (Stratagene) with LexA-SAP130, and pCMV3-SAP130 as templates. All constructs were verified by DNA sequencing. The constructs pRS-hMR and pMMTV-Luc were generous gifts from Dr. Ron M. Evans (The Salk Institute, La Jolla, CA). Plasmid M50 Super 8 × TOPFlash was a gift from Randall Moon (Addgene plasmid # 12,456; http://n2t.net/addgene:12456; RRID: Addgene_12456). Plasmid pFR-Luc with five Gal4-binding sites upstream of the minimal promoter driving the luciferase reporter gene and the NF-κB luciferase reporter construct (pNF-κB-Luc) were purchased from Stratagene. The following antibodies were purchased: HA (HA.11; Babco/Covance, Berkley, CA), HA (Biolegend, San Diego, CA), FLAG (M2; Sigma), c-Myc (9E10; GeneTex, Taiwan), GFP (JL-8; BD Biosciences Clontech), FAF1 (C1C3; GeneTex, San Antonio, TX); SAP130 (12130–1-AP, Proteintech), HDAC1 (10E2, Cell Signaling Technology), SIN3A (D9D6, Cell Signaling Technology) and actin (clone AC-74; Sigma, St. Louis, MO).

Yeast two-hybrid screening and β-Gal assay

Yeast two-hybrid library screening with full-length human FAF1 bait was performed as described previously [24]. Briefly, we transformed the L40 yeast strain with the LexA-FAF1 plasmid followed by transformation with 200 μg of the human testis cDNA library (Clontech). Yeast transformants were then selected on medium containing 5 mM 3-amino-(1,2,4) triazole (Sigma) lacking histidine, leucine, and tryptophan. Histidine protrotophic (His +) colonies were further tested for β-galactosidase activity. The plasmids from both His + and X-gal + colonies were isolated by the curing process of MC1066 bacterial strain and sequenced. The interaction strength was determined according to the appearance of blue color on X-Gal plates, or by the quantitative liquid β-galactosidase assays using lysates from three separate yeast cultures according to the instructions of the Galacto-Light Plus kit (Applied Biosystems).

Cell culture, transfection and lentivirus-based short hairpin (sh)RNA transduction

HEK293, HEK293T, HeLa, COS-1, HCT116 and L-Wnt3a cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS). NB4 cells were maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA) containing 10% FBS. The NB4 cell line was kindly gifted from Dr. Tsai-Yun Chen (National Cheng Kung University, Tainan, Taiwan). All other cell lines were obtained from the American Type Culture Collection (Manassas, VA). All cells were incubated at 37 °C in a 5% CO2 atmosphere. Plasmids were transfected using the PolyJet™ reagent (SignaGen Laboratories, Gaithersburg, MD). For stable SAP130 transfection of HEK293 cells, the plasmid constructs of pCMV3-HA-SAP130 WT or 3KA mutant were transfected into HEK293 cells and selected with hygromycin (100 μg/ml). Human FAF1 shRNA (TRCN0000004244) MISSION® shRNA Lentiviral Transduction Particles were purchased from Sigma-Aldrich (St. Louis, MO). To generate lentivirus-shRNA FAF1 knockdown cells, HEK293T, HCT116 and NB4 cells were infected at low confluence (20%) for 24 h with lentiviral supernatants diluted 1:1 with normal culture medium in the presence of 8 μg/ml of polybrene (Sigma). Forty-eight hours after infection, cells transduced by lentiviruses were selected under 2 μg/ml puromycin for 1 week and then passaged before use.

Growth curves and colony formation assays

The growth rate of cells was monitored by seeding 2 × 105 cells in 60-mm dishes containing 5% FBS. Cells were counted at 24-h intervals using a hemocytometer over a period of seven days. For the colony formation assay, cells were plated at a low density onto 60-mm dishes, and the medium was changed every 3 days. Two weeks later, the number of colonies was counted using the Sigmascan software program after staining with 2% methylene blue.

Immunoprecipitation and western blotting

For testing the protein–protein interactions in mammalian cells, the expression construct encoding HA-tagged wild-type SAP130 or SAP130 3KA mutant, along with the Flag-tagged FAF1 or myc-tagged mSin3A expression construct, was transfected into COS-1 cells. At 48 h after cotransfection, cells were solubilized in modified RIPA buffer consisting of 50 mM Tris–HCl (pH 7.8), 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.5% Nonidet-P40, 0.1% sodium deoxycholate, and a protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN). To measure the levels of sumoylation, various SAP130 point mutants or wild-type SAP130 and EGFP-SUMO1 constructs were transfected into COS-1 cells. The cells were then lysed with modified RIPA buffer containing 20 mM N-ethylmaleimide (NEM; Sigma). For detection of ubiquitinated SAP130 proteins by immunoprecipitation, HEK293 cells were co-transfected with the plasmid encoding Myc-Ubiquitin and wild-type HA-SAP130, along with plasmids encoding Flag-FAF1 or DM mutant, followed by the treatment with MG132 (5 μM) for another 6 h. Whole cell lysates were mixed with antiserum against HA (Babco/Covance) antibody, and the immunocomplexes were mixed with protein G Plus/protein A-agarose beads (Merck Millipore). After overnight incubation at 40C, the immunocomplexes were then gently washed three times with the PBS buffer followed by Western blot analysis. For Western blot analysis, immunoprecipitated molecules or total cell lysates were separated by SDS-PAGE, transferred onto nitrocellulose membranes (Amersham, GE Healthcare, Germany), blocked with 5% milk, and probed with anti-FLAG or anti-GFP or anti-Myc antibodies. Specific blot signals were visualized on an X-ray film by incubating with ECL chemiluminescence kit (Amersham Biosciences). The specific intensity of each protein band on X-ray film was measured by Image J software (NIH, USA) and results were normalized to corresponding actin band densities.

Dual-luciferase reporter assay

For reporter gene assays, cells were transfected in 6-well plates with 2 μg of DNA, including the indicated reporter constructs and SAP130 or FAF1 expression vectors, and the constitutive Renilla luciferase control reporter vector pRL-TK (Promega, Madison, WI). The total amount of plasmid per well was kept constant by adding pcDNA3 empty vector. For the experiments of MR activation, cells were treated with either vehicle or 10−6 M of aldosterone at 24 h post-transfection, and reporter gene activity was measured after an additional 24 h. To monitor NF-κB transcriptional activity, cells were treated with TNF-α (20 ng/ml) for 6 h at 36 h post-transfection. To stimulate Wnt signaling in HEK293 cells, cells were co-transfected with the TopFlash-Luc reporter, pRL-TK, and HA-FAF1 or SAP130 WT or 3KA mutant plasmids. Cells were treated with Wnt3A conditioned medium (CM) or left untreated and lysed for a luciferase assay at 48 h post-transfection. CM from cells expressing Wnt3A was prepared from L-Wnt3A cells (CRL-2647; American Type Culture Collection Manassas, VA) after 4 days and an additional harvest following 3 days in culture. CM were combined, sterile filtered and mixed 1:1 with normal media for use. Luciferase activity was determined using the dual-luciferase reporter assay system (Promega, Madison, WI).

Immunofluorescence

HeLa or COS-1 cells were transfected with HA-tagged FAF1, SAP130 WT or 3KA construct by the lipofection method. At 48 h after transfection, the cells were fixed for 10 min with 4% paraformaldehyde in phosphate-buffered saline (PBS) and then permeabilized with cooled acetone for 1 min at -20 °C. The permeabilized cells were then incubated with anti-HA monoclonal antibody alone or combined with SIN3A or HDAC1 antibody for 1 h at room temperature. Subsequently, cells were washed three times with PBS and then incubated with fluorescein isothiocyanate-conjugated anti-mouse or rabbit IgG (Jackson ImmunoResearch) alone or combined with Texas red-conjugated anti-mouse IgG at room temperature for one hour. Nuclei were stained by DAPI (4′,6′-diamidino-2-phenylindole,10 μg/ml). After washing again with PBS, the coverslips were inverted and mounted with GEL/Mount (biomeda corp) to prepare the fluorescence images for analysis with an Olympus BX51 microscope.

In vitro sumoylation assays

Expression constructs of HA-tagged wild-type or KA mutant SAP130 were transiently transfected into COS-1 cells. Cell extracts were harvested 48 h later for immunoprecipitation with anti-HA antibody and followed by in vitro sumoylation assays. In vitro sumoylation assays were performed as described previously [24]. A typical sumoylation reaction was performed in 20 μl of reaction mixture containing 150 ng of SUMO E1 recombinant proteins (LAE Biotechnology, Taichung, Taiwan), 1 μg of SUMO-1, 1 μg of Ubc9, and proteins bound to beads in a reaction buffer (2 mM ATP, 20 mM HEPES pH 7.5, and 5 mM MgCl2). The reactions were carried out at 37 °C for 2 h, and the reactions were stopped by washing with PBS for three times. Beads of samples were analyzed by SDS-PAGE followed by Western blot analysis using anti-HA antibody.

RNA extraction and real-time quantitative PCR (qPCR)

Total RNA was isolated from control, HEK293 SAP130 WT and 3KA stable cells (approximately 2 × 107) using an RNeasy mini kit (Qiagen, Valencia, CA) and then reverse-transcribed using the ThermoScript RT-PCR system (Invitrogen). The real-time qPCR analysis was performed using the SYBR Green Advantage qPCR Premix (Clontech) and C1000™ Thermal Cycler (Bio-Rad Laboratories, Hercules, CA). PCRs were performed using the following conditions for 30 cycles: 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 20 s. The following primer pairs were used. SAP130, forward (5’-CGGGTCAAAGAGGAGAAGAAA-3’) and reverse (5’-CAGCACAGAGGTGGACTTT-3’), and GAPDH, forward (5’-CCCACTCCTCCACCTTTGAC-3’) and reverse (5’- TCTCTCTTCCTCTTGTGCTCTTG-3’). Relative amounts of the SAP130 transcripts were determined using the comparative CT method and were normalized to the GAPDH housekeeping control.

cBioPortal analysis

cBioportal website (http://www.cbioportal.org/) is an open-access resource for the interactive exploration of multidimensional cancer genomics data [27, 28]. Using the cBioPortal database, the correlations between mRNA expression of SAP130 and the FAF1 in various cancer types were investigated.

Statistical analysis

Statistical analyses were performed using two-tailed Student's t-test. P values were calculated using Prism v. 3.03 (GraphPad Software, San Diego, CA).