Specimens and transfection
Chorionic villi samples used were described previously . Various human cancer cells (HeLa, HEC-1A, NCCIT, and fibrosarcoma cells) were cultured in DMEM (Gibco BRL, Rockville, MD, USA) supplemented with 10% fetal bovine serum (Gibco BRL, Rockville, MD, USA). Transfection was performed with LipofectAMINE reagent (Gibco BRL, Rockville, MD, USA) according to manufacturer's instruction.
cDNA synthesis and cloning for USP17 subfamily members
TRIzol reagent (Gibco BRL, Rockville, MD, USA) was used for total RNA isolation from obtained chorionic villi, HeLa, HEC-1A, NCCIT, and fibrosarcoma cells. cDNA synthesis from obtained total RNA was in accordance with the protocol of SuperScript Preamplification system (Gibco BRL, Rockville, MD, USA). The full-length cDNAs were amplified by RT-PCR in human chorionic villi tissues, HeLa, HEC-1A, NCCIT, and fibrosarcoma cells. Primers (Forward: 5'-CGGGATCCATGGAGGACG-3', Reverse: 5'-GGAATTCCACTGAGATCACT-3') used for isolating putative DUB genes are made on the basis of published sequences [GenBank: XM_252830]. Primers used were synthesized commercially and gel-purified prior to use (Bioneer, Daejeon, Korea). To express GST fusion proteins, pGEX-2TK-USP17K, pGEX-2TK-USP17L, pGEX-2TK-USP17M, and pGEX-2TK-USP17N were cloned by inserting each USP17 subfamily member into pGEX-2TK vector (Amersham Life Science, Uppsala, Sweden). BLAST programs on the web site established by the National Center of Biological Information  were used for similarity searches in the GenBank, and Pfam  program was used for prediction of possible motifs and domains in the protein sequences.
Site-directed mutagenesis of USP17 subfamily members
Missense mutation constructs for pGEX-2TK-USP17K (C89S), pGEX-2TK-USP17L (C89S), pGEX-2TK-USP17M (C89S), and pGEX-2TK-USP17N (C89S) were derived from each wild type construct by PCR-based site-directed mutagenesis, using a QuikChange™ Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA) according to manufacturers' instructions. The two primers; forward primer (5'-GGGAAATACCCGCTACGTGAACGCTTC-3') and reverse primer (5'-GAAGCGTTCACGTAGCGGGTATTTCC-3') were used for replacing cysteine with serine at amino acid 89 of USP17 subfamily proteins. Primers used were synthesized commercially and gel-purified prior to use (Bioneer, Daejeon, Korea).
Northern blot analysis
As described previously , Northern blot analysis was carried out to confirm the expression level of USP17 transcripts using a human tissue blot purchased from Clontech. The immobilized nucleic acids were hybridized with radiolabeled DNA probes. Probes used for this study was the 5' 500 bp and 3' 1 kb of USP17L. They were labeled by random primers with [32P]dCTP (Amersham Biosciences, Buckinghamshire, England). Hybridization was performed in a bag containing 5 × SSC, 1 × Denhardt's solution, 100 μg/ml of denatured salmon sperm DNA, 0.1% SDS and 50% formamide at 60°C and it was washed in 0.1 × SSC and 0.2% SDS, the membrane was exposed to Kodak X-ray films with an intensifying screen for 24 hours at -70°C.
Deubiquitinating enzyme assays
Deubiquitinating enzyme assays were previously described . For the in vitro deubiquitination assay, pGEX-2TK-USP17K, pGEX-2TK-USP17K (C89S), pGEX-2TK-USP17L, pGEX-2TK-USP17L (C89S), pGEX-2TK-USP17M, pGEX-2TK-USP17M (C89S), pGEX-2TK-USP17N, and pGEX-2TK-USP17N (C89S) were co-transformed into E. coli BL21 competent cells (Stratagene, La Jolla, CA, USA) with pACYC184-Ub-β-gal. Plasmid-bearing E. coli BL21 cells were grown in LB medium and induced with isopropyl-1-thio-β-D-galactopyranoside (IPTG) for 4 hours at 37°C. Cells were harvested and were lysed in cracking buffer (0.01 M phosphate [pH 7.4]. 8 M Urea, 1% SDS, and 1% β-mercaptoethanol). These whole cell lysates centrifuged for 10 min at 14,000 rpm and the supernatant was loaded onto a 7.5% SDS-PAGE gel. The immunoblotting was performed with a rabbit anti-β-gal antiserum (ICN, Costa Mesa, CA, USA) or a rabbit anti-GST antiserum (Upstate Biotechnology, Lake Placid, NY, USA).
The effect of USP17 expression on the ubiquitin-proteasome system in vivo was analyzed by transfecting pcDNA3-myc-USP17L or pcDNA3-myc-USP17L (C89S) along with pMT123-HA-ubiquitin into HEC-1A cells. After 24 hours of transfection, cells were harvested and lysed. 40 μg of total proteins was loaded in each lane of a 10% SDS-PAGE for immunoblotting analysis using an anti-HA antibody (Roche Applied Science, Mannheim, Germany). Equal loading was verified by immunoblotting against α-myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Real-time PCR analysis and the Tet-on system
After the transfection of pcDNA3-myc-USP17L, the expression of caspase 3 as a marker for apoptosis was analyzed by real-time PCR (Opticon2, MJ Research, Ramsey, MN, USA). Product specificity was examined by melting curve analysis and agarose gel electrophoresis after each real-time PCR reaction. To normalize the expression data of the genes, GAPDH was used (Forward primer: 5'-ACTGGTGCTGCCAAGGCTGT-3', Reverse primer: 5'-TCCACCACCCTGTTGCTGTA-3'). Quantification was performed using an internal cDNA standard curve. Fold changes of caspase 3 expression in USP17L-overexpressed cells were calculated relative to the levels in control cells.
For the long-term (3 weeks) observation and FACS analysis, USP17L was expressed in HeLa cells using the Tet-on system (Gibco BRL, Rockville, MD, USA) by manufacturer's instruction. Expression of USP17L was confirmed by real-time PCR analysis.
For apoptosis analysis, HeLa-Tet-on cells expressing USP17L were harvested in PBS and resuspended 100 μl of binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). 5 μl annexin V-FITC (BD Biosciences, Bedford, MA, USA) and 10 μl propidium iodide (PI, 50 μg/ml, BD Biosciences, Bedford, MA, USA) were added and incubated 15 minutes in the dark. Finally 400 μl of binding buffer was added and analyzed by FACSVantage™ SE system (BD Biosciences, Bedford, MA, USA). Cells in stage of apoptosis were differentiated depending on the location in quadrant (annexin V +, PI -; early stage) and (annexin V +, PI +; late stage) .
USP17L and USP17N were subcloned into pEGFP-C1 and pDsRed-C1 (Clontech, Palo Alto, CA, USA), respectively, and transfected into HeLa cells using ExGen500 (Fermentas, Hanover, MD, USA). After 48 hours, the localization of GFP-tagged USP17L and RFP-tagged USP17N was observed using a confocal microscopy (Radiance 2100 2Q, Bio-Rad, Hercules, CA, USA). In order to observe the morphology of nuclei for HeLa cells, DAPI staining was performed. After the fixation, 5 μl of DAPI staining solution (Roche Applied Science, Mannheim, Germany) was added in wells containing HeLa cells, which express either GFP-tagged USP17L or RFP-tagged USP17N. Stained cells were observed using Nikon Eclipse E600 microscopy.
Hyaluronan- and RNA-binding assays
These assays were previously described . 9 μg of pcDNA3-myc-USP17L or pcDNA3-myc-USP17N was transiently transfected into 1 × 106 HeLa cells in 100Φ dish by ExGen500 transfection reagent (Fermentas, Hanover, MD, USA). The supernatant from lysates was divided into 100 μl aliquots in Eppendorf tube and incubated at room temperature with 50 μg of hyaluronan (H5388, Sigma, St. Louis, MO, USA). For RNA binding assay, this procedure was repeated with using total RNA of HeLa cells instead of hyaluronan.