Chemicals, cell lines and small interfering RNA (siRNA)
17β-estradiol (E2) was purchased from Sigma (St Louis, MO, USA). Steroid-deprived serum was prepared as described previously . Phenol-red free Dulbecco's modified Eagle's medium (DMEM) was from the Institute of Basic Medicine, Beijing Union Hospital (Beijing). MCF-7 cells were originally obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured according to ATCC instructions. Duplexes of K18 specific siRNAs 361 (sense strand, 5'-GACCATGCAAAGCCTGAAC-3'), 609 (sense strand, 5'-GAGTCAAGTATGAGACAGA-3') and 908 (sense strand, 5'-GAGGAGCTAGACAAGTACT-3') were chemically synthesized by Shanghai GeneChem Co. (Shanghai). The unrelated siRNA sequence (sense strand, 5'-GACGAACGTGTCACGTATC-3') was used as a control.
The pcDNA3.1-LRP16 and the pcDNA3-Flag plasmid were described previously . The human ERα expression vector pSG5-hERα was kindly provided by Dr. Hajime Nawata (Kyushu University, Japan). The reporter 3× ERE-TATA-Luc was provided by Prof. Donald P. McDonnell (Duke University Medical Center, Durham, NC, USA). The LRP16-GFP fusion expression vector was constructed by inserting the full-length LRP16 cDNA at the Kpn I and BamH I sites of the pEGFP-N1 vector. The yeast expression plasmid pGBKT7-LRP16 (Gal4 BD:bait gene fusion) was generated by inserting the full-length LRP16 cDNA in-frame at the Eco RI site of pGBKT7. To generate the GST-LRP16 fusion plasmid and its mutants GST-LRP16-N (1-160) and GST-LRP16-C (161-324) the corresponding fragments were PCR-amplified and inserted at the Eco RI/Hin dIII sites of plasmid pGEX-6p-1 (Amersham Biosciences, Freiburg, Germany). The full-length coding region of human K18 was amplified from GAL4 AD:K18 (pGADT7-K18) and then cloned at the BamH I/EcoR I sites of pcDNA3.1. To generate K18 deletion mutants K18-N (amino acids 1-150), K18-F (80-375), K18-C1 (301-430) and K18-C2 (390-430), the corresponding fragments were PCR-amplified and inserted at the Eco RI/Xho I sites of the pcDNA3.1 or pcDNA3-Flag vectors.
Generation of the cDNA library and yeast two-hybrid screening
Total RNA from MCF-7 cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and a cDNA library was generated using the BD SMART™ kit (Clontech, Palo Alto, CA, USA) according to the manufacturer's instructions. Yeast two-hybrid screening for the identification of LRP16-interacting proteins involved the MATCHMARKER two-hybrid system 3 kit (Clontech) according to the manufacturer's instructions.
GST pull-down assay
GST and GST fusion proteins were prepared as described previously . 35S-labeled proteins were produced with use of a TNT-coupled in vitro transcription and translation system (Promega Corporation, Madison, WI, USA) with the expression vector K18 and its derivatives in pcDNA3.1.
Extraction of cytoplasmic/nuclear proteins, co-immunoprecipitation (CoIP) and immunoblotting
Cells were cultured in 10 cm dishes and transfected with expression vectors or siRNA duplexes. 48 h after transfection, cells were harvested and lysed for CoIP or immunoblotting assays. Extraction of total, cytoplasmic, or nuclear proteins employed the ReadyPrep™ protein extraction kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the instruction manual supplied by the manufacturer. For CoIP assays, cells were lysed in 500 μl lysis buffer (20 mM Tris [pH 7.4], 50 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.5% SDS, 0.5% deoxycholate, and protease inhibitors). To efficiently solubilize keratins, cells were treated with 2% Empigen BB (Sigma) as described previously . Lysate aliquots (500 μg; 1 μg/μl) were precleared with 50 μl of protein A-Sepharose beads (Upstate Biotechnology, Lake Placid, NY, USA) for 2 h at 4°C. Appropriate amounts of rabbit anti-LRP16, rabbit anti-Flag (Sigma), rabbit anti-ERα(Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit nonspecific IgG (Clontech) was then added and incubated overnight at 4°C. Preblocked agarose beads (100 μl) were then added to the antibody/lysate mixture and incubation was continued for a further 2 h at 4°C. After washing (3×), bound proteins were eluted in SDS sample buffer, resolved by SDS-PAGE, and analyzed by immunoblotting. The rabbit and mouse anti-LRP16 antibodies were as described previously . Antibodies used for immunoblotting were mouse anti-K18 (Abgent, San Diego, CA, USA), mouse monoclonal anti-Flag (Sigma), rabbit anti-ERα, mouse anti-Sp1 and rabbit anti-β-actin (Santa Cruz Biotechnology).
Quantitative analysis of LRP16-GFP subcellular localization
MCF-7 cells were grown in 35 mm culture dishes and cotransfected with LRP16-GFP and K18 or pcDNA3 empty vector. 24 h after transfection cells were fixed with 3% formaldehyde (15 min) and nuclei were counterstained with 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI). Cells were visualized under an inverted fluorescence microscope (IX-71; Olympus) equipped with a digital camera. The proportion of cells displaying LRP16-GFP in the nucleus was determined by counting at least 500 cells from each plate. The means and SEM were calculated from 3 separate plates from 3 independent experiments.
MCF-7 cells were cultured in phenol-red free media stripped of steroids for at least 3 days and were then seeded into 35 mm culture dishes. Cells at 50% confluence were cotransfected by use of Superfect (Qiagen, Valencia, CA, USA). Cells were cotransfected with 0.5 μg of the reporter construct and 0.25 μg of ERα- and/or 0.5 μg of K18- or LRP16-expression vectors. Cotransfaction with plasmid pRL-SV40 (1 ng/per well) was used to control for transfection efficiency. Total DNA was adjusted to 2 μg per well with pcDNA3.1 empty vector. 36 h after transfection cells were treated with or without E2 (100 nM), cultured for a further 6 h, and cell extracts were prepared and relative luciferase activities were measured as described previously . For knockdown experiments, 1 μg of siRNA duplexes, 0.5 μg of the reporter construct, 0.25 μg of the ERα-expression construct and 1 ng of pRL-SV40 were cotransfected using Lipofectamine 2000 according to the manufacturer's recommendations (Invitrogen). The total amount of nucleotides was adjusted to 4 μg per well with pcDNA3.1 empty vector. 42 h after transfection cells were treated with or without E2 (100 nM) and cultured for a further 6 h, harvested, and the relative luciferase activity was measured as described previously .
Quantitative RT-PCR (qPCR)
Total RNA was extracted with use of TRIzol reagent (Invitrogen) and qPCR analysis was performed as described previously . cDNA was prepared by use of Superscript II RNase H- reverse transcriptase (Invitrogen) and 1-2 μg total RNA. The optical density was measured and equal amounts of cDNA were used in a normalization reaction with primers for HPRT. Oligonucleotide primers were as follows: HPRT sense, 5'-TTGCTCGAGATGTGATGAAAGGA-3'; HPRT antisense, 5'-TTCCAGTTAAAGTTGAGAGATCA-3'; pS2 sense, 5'-ATGGCCACCATGGAGAACAA-3'; pS2 antisense, 5'-TAAAACAGTGGCTCCTGGCG-3'; cyclinD1 sense, 5'-CTGGCCATGAACTACCTGGA-3'; cyclinD1 antisense, 5'-GTCACACTTGATCACTCTGG-3'; c-Myc sense, 5'-GACTATCCTGCTGCCAAGAG; and c-Myc antisense, 5'-TCGCCTCTTGACATTCTCCT-3'. Reactions were run on a LightCycler (Roche, Indianapolis, IN, USA). Experiments were performed in triplicate and repeated at least 3 times.
Chromatin immunoprecipitation (ChIP) assays
MCF-7 cells (1 × 106) were grown in 10 cm tissue culture plates in phenol-red free DMEM supplemented with 10% (v/v) steroid-depleted FBS. After 24 h the cells were transfected with 10 μg of pcDNA3.1-K18 or empty vector DNA using the Superfect reagent. 40 h later, transfected cells were treated with E2 (100 nM) for 1 h and were then analyzed by ChIP. Briefly, immunoprecipitation was carried out overnight at 4°C with ERα (Santa Cruz Biotechnology) antibody or nonspecific IgG antibody. DNA fragments were purified with use of a QIAquick Spin Kit (Qiagen). The presence of target gene promoter sequences in both input and recovered DNA immunocomplexes was detected by PCR. The promoter region (nt -353 to -30) of the pS2 gene was amplified.
G1/S checkpoint assay
MCF-7 cells were cultured in phenol-red free medium stripped of steroids for at least 3 days, and were then seeded in 35 mm culture dishes and cotransfected with plasmids pcDNA3.1, pcDNA3.1-K18 and/or pcDNA3.1-LRP16 or K18-siRNA/control-siRNA. A vector expressing enhanced green fluorescent protein (EGFP) was used to identify transfected cells as described previously . After 36 h, cells were treated with or without E2 (100 nM) for a further 12 and were then labeled with 10 μM BrdU for 2 h. Immunostaining was performed using anti-BrdU antibody (Becton Dickinson, Franklin Lakes, NJ, USA). The ratios of BrdU and EGFP double-positive cells to EGFP-positive cells were determined using an Olympus fluorescence microscope. At least 350 cells from each plate were counted. The means and SEM were calculated from 3 separate plates from 3 independent experiments.
Results were expressed as the means ± standard error of the mean (SEM). Statistical analysis involved use of Statview 5.0 software. P < 0.05 was considered statistically significant.