Doxycycline-inducible U2OS C3 cell culture, siRNA transfection, and synchronization.
We previously reported on the generation of an osteosarcoma U2OS clone C3 cell line (U2OS C3 cells) that allowed Dox-inducible expression of the GFP-FoxM1b fusion protein (27
). U2OS C3 cells were maintained as a monolayer in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mM l
-glutamine, and 50 μg/ml of hygromycin B (Invitrogen). Dharmacon Research synthesized two 21-nucleotide siRNA duplexes specific to human FoxM1 mRNA, named siFoxM1 #1 (CAACAGGAGUCUAAUCAAG) and siFoxM1 #2 (GGACCACUUUCCCUACUUU), and human p27Kip1
siRNA duplex (GUACGAGUGGCAAGAGGUGUU), containing symmetric 2-uracil (U) 3′ overhangs. Human Aurora B siRNA duplex was purchased from Cell Signaling Technology (Beverly, MA). These siRNA duplexes were transfected into U2OS C3 cells using Lipofectamine 2000 reagent (Invitrogen) in serum-free tissue culture medium following the manufacturer's protocol. Four hours after transfection, the cells were fed with complete DMEM containing 10% FCS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mM l
-glutamine, and 50 μg/ml of hygromycin B. For induced expression of the GFP-FoxM1b fusion protein, we added complete DMEM containing 10% FCS and 1 μg/ml of Dox (Sigma) 4 hours following FoxM1 siRNA transfection. U2OS C3 or U2OS cells were harvested at 48 h after FoxM1 siRNA transfection for total RNA or harvested at 72 h after FoxM1 siRNA transfection to prepare protein extracts for Western blot analysis, flow cytometry, or immunofluorescent staining. For CENPA immunofluorescent staining of mitotic synchronized cells, at 72 h after FoxM1 siRNA 2 transfection, we synchronized U2OS cells at early stages of mitosis with 100 ng/ml nocodazole (Sigma) for 24 h and then released them for 1 h to allow progression into metaphase.
Cell growth rate of FoxM1-depleted U2OS C3 cells by siRNA transfection.
U2OS cells were transfected with siFoxM1 #2 or siRNA p27Kip1 duplexes and then incubated for 2 days to allow siRNA silencing of expression. The FoxM1- or p27Kip1-depleted U2OS cells were then trypsinized, and the cell growth rate was determined in triplicate at 3, 4, 5, or 6 days after siRNA transfection. To determine cell number, U2OS cells were trypsinized and removed from the plate, nonviable cells were identified by 3 min of staining with 0.4% trypan blue (Sigma), and the number of unstained U2OS C3 cells was counted using a hemocytometer. We plotted cell number ± standard deviation (SD) versus days after siRNA transfection. Statistical analysis of experiment data was performed with Microsoft Excel tools.
Generation and growth analysis of Foxm1−/−, Foxm1+/−, and Foxm1+/+ MEFs. Foxm1+/−
mice containing an inactivated Foxm1-targeted allele that deleted essential Foxm1
exons 4 through 7, which encode the Foxm1 DNA binding domain and C-terminal transcriptional activation domain, were described previously (36
). We bred Foxm1+/−
mice to generate 13.5-day embryos with Foxm1+/+
(wild type), Foxm1+/−
(heterozygous), or Foxm1−/−
(knockout) genotypes for isolation of MEFs. To isolate MEFs from the embryos, the liver, heart, and head were removed from the embryos and then the remaining embryo was digested with 0.25% trypsin in 2.21 mM EDTA (Cellgro, Herndon, VA) to isolate single-cell suspensions of MEFs using standard procedures described by Hogan et al. (23
). The heart tissue was used to generate DNA for PCR genotyping of the embryos as described previously (36
). The MEFs were grown in DMEM supplemented with 10% FCS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mM l
-glutamine, 0.1 mM MEM nonessential amino acids, and 55 mM 2-mercaptoethanol in a humidified 9% CO2
incubator under conditions described by Zindy et al. (76
). To measure cell population doublings, MEFs were trypsinized and then 3 × 105
cells were replated and fed with DMEM containing 10% FCS. Following 3 days of growth at 37°C and 9% CO2
, we counted the number of cells as described above. Population doubling during each passage was calculated according to the formula log[(cell number after 3 days of growth)/(3 × 105
Flow cytometry assays to determine cell cycle profiles.
U2OS C3 cells were transfected with 100 nM of either siFoxM1 #2, p27Kip1, or Aurora B kinase siRNA duplexes, and then 72 h after transfection the cells were subjected to flow cytometry to analyze their cell cycle profile. Wild-type (WT) Foxm1+/+, heterozygous Foxm1+/−, or knockout Foxm1−/− MEFs were trypsinized at passage 4 to analyze their cell cycle profile by flow cytometry. For flow cytometry, cells were trypsinized and fixed in 70% ethanol for 2 h at 4°C. Cells were incubated with 40 μg/ml propidium iodide and 100 μg/ml RNase A (Sigma) in phosphate-buffered saline (PBS) for 1 h at 37°C. After washing, cells were resuspended in PBS for further analysis. Data were acquired using a Beckman Coulter EPICS Elite ESP apparatus (Hialeah, FL) and then analyzed using Multicycle AV (Phoenix Flow Systems, San Diego, CA). The flow cytometry and analysis were performed in the Research Resource Center at the University of Illinois at Chicago.
Description of antibodies used for Western blot analysis to determine protein expression levels.
To prepare protein extracts, U2OS cells depleted of FoxM1 or Aurora B kinase or Foxm1+/+
, or Foxm1−/−
MEFs were harvested in ice-cold PBS, pelleted by centrifugation, and used to make whole-cell protein extracts using the NP-40 lysis buffer as described previously (42
). Cytoplasmic and nuclear protein extracts were made using a nuclear/cytosol fractionation kit (K266-100; BioVision) following protocols provided by the manufacturer. Protein concentrations were determined by the Bradford method with the Bio-Rad protein assay reagent. Equal amounts of proteins in whole-cell extracts or nuclear and cytoplasmic extracts from each set of experiments were fractionated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membrane (Bio-Rad). The membrane was subjected to Western blot analysis with antibodies against proteins of interest as described previously (27
). The signals from the primary antibody were amplified by horseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG; Bio-Rad, Hercules, CA) and detected with enhanced chemiluminescence (ECL Plus; Amersham Pharmacia Biotech, Piscataway, NJ).
The following commercially available antibodies and dilutions were used for Western blotting: mouse anti-Plk-1 (F-8; 1:500); mouse anti-Cdc25A (F-6; 1:300); rabbit anti-Cdk2 (M2; 1:1,500), and mouse anti-cyclin A (H-432; 1:2,000) (Santa Cruz Biotechnology, Inc.); rabbit anti-INCENP (1:5,000), mouse anti-β-actin (AC-15; 1:5,000) (Sigma); mouse anti-Cdc25B (1:250), mouse anti-Aurora B kinase/AIM-1 (1:250), mouse anti-Kip1/p27 (1:3,000), mouse anti-Cip1/p21 (1:3,000), and mouse anti-human cyclin B1 antibody (GNS-11; 1:500) (BD Biosciences); mouse anti-GFP (JL-8; 1:1,000; Clontech, Franklin Lakes, NJ); rabbit anti-CENPA (1:200; Upstate), rabbit anti-Aurora A/AIK (1:1,000), and mouse antisurvivin (6E4; 1:500) (Cell Signaling Technology); and mouse anti-p45/Skp2 (2B12; 1:500) and rabbit anti-Cks1 (C-term; 1:500) (Zymed, South San Francisco, CA). The rabbit anti-Cullin 4A (Cul4A) antibody (1:1,000) was a gift from P. Raychaudhuri (University of Illinois at Chicago).
Generating rabbit antisera specific to the human C-terminal FoxM1b protein region.
We cloned the human FoxM1b 365-748 amino acid protein into a His-tagged expression vector. The His-tagged FoxM1b 365-748 amino acid protein was expressed in Escherichia coli and affinity purified by nickel chromatography following the manufacturer's protocol (Invitrogen). To generate a rabbit FoxM1 antibody, we provided Genemed Synthesis, Inc. (South San Francisco, CA) with affinity-purified His-tagged FoxM1b 365-748 amino acid protein as an antigen to immunize two rabbits, and the subsequent antibody production consisted of initial immunization followed by six boosts with the His-tagged FoxM1b 365-748 antigen. For Western blot analysis we used the Genemed-generated rabbit anti-FoxM1 antibody at a 1:5,000 dilution using procedures described above.
Procedure for immunofluorescent staining of U2OS cells and MEFs.
U2OS C3 cells or MEFs were fixed with 10% buffered formalin (Fisher) for 20 min at room temperature, rinsed with PBS, and permeabilized with PBS supplemented with 1% bovine serum albumin (BSA; Sigma) and 0.2% Triton X-100 (Fisher). After washing in PBS with 1% BSA, proteins of interest were visualized by staining cells with specific antibodies in PBS containing 0.5% BSA at 25°C for 16 h. The following commercially available antibodies and dilutions were used for immunofluorescent staining: mouse anti-Aurora B kinase/AIM-1 (1:25), mouse anti-p27Kip1 (1:200), and mouse anti-p21Cip1 (1:200) (BD Transduction Laboratories); rabbit anti-CENPA (1:75) and rabbit anti-phospho-histone H3 (Ser10; 1:100) (Upstate); mouse anti-α-tubulin (1:1,000; Sigma); anti-P16Ink4A (1:100; Santa Cruz); and anti-p19ARF (1:100; Abcam). After being washed with PBS, cells were incubated with tetramethyl rhodamine isocyanate-conjugated polyclonal anti-mouse immunoglobulins (1:100) or fluorescein isothiocyanate-conjugated polyclonal anti-mouse immunoglobulins (1:100; DakoCytomation, Denmark) or Texas Red-conjugated anti-mouse IgG antibody (1:150; Vector Laboratories) in PBS containing 0.5% BSA at 25°C for 30 min. The slides were washed with PBS, and coverglasses were mounted with Vectashield mounting medium with 4′,6′-diamidino-2-phenylindole (DAPI; H-1200; Vector Laboratories). Immunofluorescence with primary antibodies followed by secondary antibodies conjugated to either tetramethyl rhodamine isocyanate or fluorescein isothiocyanate or Texas Red was detected using an Axioplan 2 microscope (Carl Zeiss).
Procedure for senescence-associated β-galactosidase staining of MEFs.
In situ SA-β-Gal activity was detected as described elsewhere (55
) with minor modifications. Passage 3 Foxm1+/+
, or Foxm1−/−
MEFs were washed with PBS and fixed with 2% formaldehyde, 0.2% glutaraldehyde in PBS for 15 min at room temperature, washed twice with PBS, and stained for 16 h at 37°C with 1 mg/ml 5-bromo-4-chloro- 3-indolyl-β-galactoside (X-Gal) (pH 6.0) in 40 mM citric acid-sodium phosphate buffer containing 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM MgCl2
, and 150 mM NaCl. Micrographs of β-galactosidase-stained MEFs were taken at 200× magnification using an Axioplan 2 microscope (Carl Zeiss).
Primers used for real-time reverse transcriptase PCR (RT-PCR) to determine mRNA expression levels.
U2OS cells or MEFs were harvested at 48 h following siRNA transfection for preparation of total RNA using RNA-STAT-60 (Tel-Test B Inc., Friendswood, TX). Following DNase I (RNase free; New England BioLabs) digestion of total RNA to remove contaminating genomic DNA, we used the Bio-Rad cDNA synthesis kit containing both oligo(dT) and random hexamer primers to synthesize cDNA from 10 μg of total RNA. The following reaction mixture was used for all PCR samples: 1× IQ SybrGreen supermix (Bio-Rad, Carlsbad, CA), 100 to 200 nM of each primer, and 2.5 μl of cDNA in a 25-μl total volume. Reactions were amplified and analyzed in triplicate using a MyiQ single-color real-time PCR detection system (Bio-Rad, Carlsbad, CA).
The following sense (S) and antisense (AS) primer sequences and annealing temperatures (Ta) were used to amplify and measure the amount of human mRNA by real-time RT-PCR: FoxM1-S, 5′-GGA GGA AAT GCC ACA CTT AGC G-3′, and FoxM1-AS, 5′-TAG GAC TTC TTG GGT CTT GGG GTG-3′ (Ta, 55.7°C); CENPA-S, 5′-CTT CCT CCC ATC AAC ACA GTC G-3′, and CENPA-AS, 5′-TGC TTC TGC TGC CTC TTG TAG G-3′ (Ta, 54°C); survivin-S, 5′-TCA AGG ACC ACC GCA TCT CTA-3′, and survivin-AS, 5′-TGA AGC AGA AGA AAC ACT GGG C-3′ (Ta, 61°C); CENPB-S, 5′-ATT CAG ACA GTG AGG AAG AGG ACG-3′, and CENPB-AS, 5′-CAT CAA TGG GGA AGG AGG TCA G-3′ (Ta, 58°C); Skp2-S, 5′-GGT GTT TGT AAG AGG TGG TAT CGC-3′, and Skp2-AS, 5′-CAC GAA AAGGGC TGA AATGTT C-3′ (Ta, 62°C); Cks1-S, 5′-GAA TGG AGG AAT CTT GGC GTT C-3′, and Cks1-AS, 5′-TCT TTG GTT TCT TGG GTA GTG GG-3′ (Ta, 55.7°C); KPC1-S, 5′-CCT GAA CCG CAT CTT TTC CTC-3′, and KPC1-AS, 5′-CTC ATT GTC CAC CTG TAG CAA CTG-3′ (Ta, 62.0°C); KPC2-S, 5′-GCC GTA TTT TTA GCA TCT GAC AGG-3′, and KPC2-AS, 5′-TTC AAG CAG CAA CTC AAG AGC C-3′ (Ta, 60.1°C); JAB1-S, 5′-ATC GGG AGG CAA CTT GGA AG-3′, and JAB1-AS, 5′-GCA TTT ACT CGG GTT TCA GTG-3′ (Ta, 61.3°C). These real-time RT-PCR RNA levels were normalized to human cyclophilin mRNA levels, and these primers are as follows: cyclophilin-S, 5′-GCA GAC AAG GTC CCA AAG ACA G-3′, and cyclophilin-AS, 5′-CAC CCT GAC ACA TAA ACC CTG G-3′ (Ta, 55.7°C).
The following sense and antisense primer sequences and annealing temperatures were used to amplify and measure the amount of mouse mRNA by real-time RT-PCR: FoxM1-S, 5′-CAC TTG GAT TGA GGA CCA CTT −3′, and FoxM1-AS, 5′-GTC GTT TCT GCT GTG ATT CC-3′ (Ta, 57.5°C); CENPA-S, 5′-AGC CGT GGT GTG TTT T-3′, and CENPA-AS, 5′-TCG GAT TCT CCT GGT CAA CT-3′ (Ta, 57.5°C); Cdc25B-S, 5′-CCC TTC CCT GTT TTC CTT TC-3′, and Cdc25B-AS, 5′-ACA CAC ACT CCT GCC ATA GG-3′ (Ta, 61.7°C); Skp2-S, 5′-GTA TGT TAG GGA ACC ATT TGC GAG-3′, and Skp2-AS, 5′-TTA GAA GGG CAC TTG GAA GAG TT-3′ (Ta, 55.7°C); Cks1-S, 5′-GAC CTC AAA GCC CTC GTG T-3′, and Cks1-AS, 5′-TGA AAC ATA AAT CCA TAA GTC ATC A-3′ (Ta, 55°C); p19ARF-S, 5′-AGA GCG GGG ACA TCA AGA C-3′, and p19ARF-AS, 5′-CTG AGG CCG GAT TTA GCT C-3 (Ta, 58.7°C). These real-time RT-PCR RNA levels were normalized to mouse cyclophilin mRNA levels, and these primers were as follows: cyclophilin-S, 5′-GGC AAA TGC TGG ACC AAA CAC-3′, and cyclophilin-AS, 5′-TTC CTG GAC CCA AAA CGC TC-3′ (Ta, 57.5°C).
Cdk2 and Cdk1 immunoprecipitation kinase reactions.
To measure the amount of Cdk2-cyclin E/A or Cdk1-cyclin B activity, FoxM1-depleted or untreated U2OS cells were lysed in NP-40 lysis buffer (42
), and 500 μg of total cellular protein extract was immunoprecipitated (IP) using a monoclonal antibody for Cdk2 or Cdk1 (Santa Cruz) for 16 h with gentle rocking at 4°C. The antibody-antigen complex was IP with 25% protein A-Sepharose for 2 h at 4°C. Immunoprecipitates were then washed twice in lysis buffer followed by an additional two washes in ADBI kinase assay buffer (42
). We used the Cdk2 or Cdk1 IP protein complexes for kinase reactions with 10 μg of the Cdk2 substrate RB amino acids 773 to 928 fusion protein or Cdk1 substrate histone H1 protein (Upstate) and radioactively labeled with [γ-32
P]ATP. Kinase reactions were performed for 30 min at 30°C followed by 15% SDS-PAGE. The gel was then fixed in 10% methanol and 10% acetic acid for 4 h, during which we changed the wash three times. The fixed SDS-PAGE gel was dried for 45 min, the radioactively labeled RB or histone H1 protein was visualized by scanning with the Storm 860 PhosphorImager, and phosphorylated bands were quantitated with the ImageQuant program (Amersham-Pharmacia Biotech).
Transfection of Aurora B kinase promoter-luciferase constructs into U2OS cells for dual luciferase assays.
We used PCR of human U2OS genomic DNA to amplify −749 bp of the human Aurora B promoter region. This PCR-amplified promoter region was cloned in the correct orientation in the pGL3-Basic Luciferase reporter plasmid (Promega). The following PCR primers were used to amplify the Aurora B kinase promoter region: forward, 5′-CGGAATACAAAGTCTAGA-3′; reverse, 5′-GTCCAAGGCACTGCTACT-3′. The Aurora B kinase promoter region was confirmed by DNA sequencing (University of Chicago Sequencing Facility).
We used Fugene 6 reagent (Roche) to transfect U2OS cells with 200 ng of either cytomegalovirus (CMV) FoxM1B expression construct or CMV empty vector with 1.5 μg of −749 bp Aurora B promoter luciferase reporter with 10 ng of CMV-Renilla
luciferase, which served as an internal control. Twenty-four hours after transfection, cells were harvested and protein extracts were prepared for dual luciferase assays (Promega) as described previously, and luciferase levels were normalized to Renilla
luciferase activity (42
). Promoter expression was expressed as the fold induction of transcriptional activity by the FoxM1b expression vector ± the SD, where promoter activity resulting from transfection with CMV empty vector was set at 1. Experiments were performed in triplicate, and statistical analysis was performed with Microsoft Excel tools.
FoxM1-depleted or untreated U2OS cells were processed for ChIP assay 3 days after siRNA transfection using published methods with additional modifications (71
). Briefly, FoxM1-depleted or untreated U2OS cells were cross-linked in situ by addition of 37% formaldehyde (Fisher Scientific) to a final concentration of 1% (wt/vol) and incubated at 25°C for 10 min with gentle swirling. The cross-linking reaction was stopped by the addition of 2.5 M glycine to a final concentration of 0.125 M followed by an additional 5 min of gentle swirling. Cells were washed once with 4°C sterile PBS and then collected by adding 1 ml of 4°C sterile PBS containing protease inhibitors (Roche, Mannheim, Germany). Cells were scraped from the dish with a razor blade and transferred into an Eppendorf tube, which was centrifuged at 2,000 × g
for 10 min. The cell pellet was then resuspended in a 2× pellet volume of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1) and placed on ice for 10 min.
The resulting extract was sonicated using a Misonix 600W sonicator (Misonix Inc., Farmingdale, NY) fitted with a 3-mm stepped microtip for 10 pulses of 15 seconds at a power setting of 30%. Between each pulse, the extract was incubated on ice for 1 min. At this stage, the processing of all experimental samples and total input was carried out according to the Upstate Cell ChIP assay protocol (catalog no. 17-295; Lake Placid, NY). For the immunoprecipitation, specific amounts of antibody as indicated were added to the precleared and clarified sample, which was incubated at 4°C with rotation for 12 to 16 h and washed according to the Upstate ChIP assay protocol. The following antibodies were used in the indicated amounts: 10, 25, or 50 μl of rabbit antiserum specific for FoxM1 protein (amino acids 365 to 748), 2 μg of rabbit serum (Vector Laboratories), and 2 μg of either rabbit CBP antibody (sc-369 [A-22]; Santa Cruz Biotechnology) or RNA polymerase II antibody (sc-899 [N-20]; Santa Cruz Biotechnology, Santa Cruz, CA). Cross-links were reversed on all samples, including 20% input, by addition of 100 μl TE (1 mM EDTA, 10 mM Tris-HCl, pH 7.4) containing 10 μg of RNase A and then incubated for 15 min at 25°C. Proteinase K (10 μg) and NaCl (4 μl of 5 M solution) were then added, and samples were digested for 16 h at 65°C. DNA was extracted from the digested samples using PCR purification columns following the manufacturer's instructions (QIAGEN, Maryland). We then used 2.5 μl of this ChIP DNA sample in the subsequent 25-μl real-time PCR mixture. The total input sample was diluted 1:10, and 2.5 μl was used for real-time PCR (10% total input).
PCR primers and reaction conditions for ChIP assay.
The primers used to amplify the following human gene promoter fragments are annotated with the binding position upstream of the transcription start site, annealing temperature, and whether in the sense or antisense orientation: Cdc25B −92S, 5′-AAG AGC CCA TCA GTT CCG CTT G-3′, and Cdc25B +120AS, 5′-CCC ATT TTA CAG ACC TGG ACG C-3′ (Ta, 62°C); Aurora B kinase (AurkB) −856S, 5′-GCA ACG AAA GGT CTA TTG GTG G-3′, and AurkB −611AS, 5′-TCT AAC TTC TCT GCC CGA TGG AG-3′ (Ta, 58°C); survivin −1531S, 5′-GGA GGA AGA AGC AGA GAG TGA ATG-3′, and survivin −1373AS, 5′-CTG GGA TTA CAG ATG TGA GCC AC-3′ (Ta, 65°C); CENPA −6695S, 5′-CCT TGG TGT TAT GCT CTG GGA AG-3′, and CENPA −6607AS, 5′-GGG CTG TTA CTG TTT TCT CAG GTT G-3′ (Ta, 60°C); CENPB −1036S, 5′-CCC AGA AAG TGA CAC ACC AAG ATG-3′, and CENPB −817AS, 5′-GGT AAG TAT CAT TGC CAA CAC GC-3′ (Ta, 60°C); Skp2 −7554S, 5′-AAT TAG CCA GGT GTG GTA GCG G-3′, and Skp2 −7428AS, 5′-CAG GCT TCA GTG TAA TGG CAC G-3′ (Ta, 62°C); Cks1 −213S, 5′-GTG AGA ACT GCC CTC CAA TAA GG-3′, and Cks1 −64AS, 5′-GTG AGA ACT GCC CTC CAA TAA GG-3′ (Ta, 64°C); transthyretin (TTR) −308S, 5′-TTA GTG CAC GCA GTC ACA CA-3′, and TTR −54AS, 5′-GCT TAT CCC TGC CAA TCT GAC TG-3′ (Ta, 62°C). The following reaction mixture was used for all PCR samples: 1× IQ SybrGreen Supermix (Bio-Rad, Carlsbad, CA), 100 nM of each primer, and 2.5 μl of each purified ChIP extract in a 25-μl total volume. Reactions were amplified and analyzed in triplicate using a MyiQ single-color real-time PCR detection system (Bio-Rad, Carlsbad, CA). Normalization was carried out using the ΔΔCt method. Briefly, IP samples and total input threshold cycles (Ct) for each treatment were subtracted from the Ct of the corresponding serum control IP (rabbit serum). The resulting corrected value for the total input was then subtracted from the corrected experimental IP value (ΔΔCt), and these values were raised to the power of 2 (2ΔΔCt). These values were then expressed as a relative promoter binding ± SD.
We used the Microsoft Excel program to calculate the SD and statistically significant differences between samples using the Student t test. The asterisks in each graph indicate statistically significant changes, with P values calculated by the Student t test. P values of <0.05 were considered statistically significant.