Cell culturing and TGF-β1 stimulation
Human embryonic lung fibroblasts (HFL-1) (ATCC, Manassas, VA, USA) were sub-cultured in Eagles minimum essential medium (EMEM) supplemented with 1% glutamine and 10% fetal calf serum (FCS) and PEST. Before experiments cells were grown to confluency and were starved overnight in Dulbecco’s modified Eagle medium (DMEM) with 0.4% FCS. Cells were washed in PBS and were then incubated with or without TGF-β1 (10 ng/mL) (R&D Systems Abingdon, UK) for the time-points indicated in the figures. Experiments were performed in passage 17–22.
Cells were grown under standardized conditions with or without TGF-β1 (10 ng/mL) for 6, 24, or 48 h and whole cell lysates were prepared using lysis buffer (50 mM Tris–HCl, 500 mM NaCl, 1% NP-40, 10% glycerol, 10 mM MgCl2, pH 7.4) containing the protein inhibitor cocktail complete mini (1 mM PMSF, 1 μg/mL Aprotinin, 1 μg/mL Pepstatin, 1 μg/mL Leopeptin, Roche, Manheim, Germany). Samples were solubilized in Laemmli’s buffer and equal amounts of total protein (10 μg) were loaded and separated by electrophoresis on 4–12% Bis-Tris Gels (Invitrogen, Gibro, Carlsbad, CA, USA). The proteins were blotted to PVDF membranes (Immobilon-P Transfer Membrane, Millipore Corporation, Billerica, MA, USA). Membranes were incubated with antibodies against: a-SMA (Abcam, Cambridge, UK), prolyl 4-hydroxylase (Acris antibodies, Hiddenhausen, Germany), fibronectin (Dako, Glostrup, Denmark), EDA-fibronectin (Abcam, Cambridge, UK), GAPDH (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), Phospho-SR proteins (Zymed Laboratories, San Francisco, CA, USA), and SRp20 (Zymed Laboratories, San Francisco, CA, USA). Bound antibodies were visualized by peroxidase-conjugated secondary antibodies and enhanced luminescence (AmershamTM western blotting Detection Reagents, GE Healthcare, Uppsala, Sweden) with exception for the blot against phospho-SR proteins where Dy-light 800 nm conjugated secondary antibodies (Cell Signaling Technology Inc., Boston, MA, USA) was used. The luminescence/fluorescence signal was detected on Odyssey® FC imaging system (LI-COR Biosciences, Lincoln, NE, USA). Exposure times were standardized so that all samples were treated the same way for each antibody. Individual bands were quantified with densitometry using the Quantity One software version 4.6.1 (BIORAD Laboratories, Hercules, CA, USA). Data are based on four individual sets of experiments.
Fibroblasts (7000/well) were grown overnight on chamber slides and were then incubated with or without TGF-β1 24 and 48 h. Cells were then fixed in 4% formaldehyde for 15 min and permeabilized with 0.1% Triton X for 30 min. After blocking in 2% BSA-TBS containing 5% goat serum (Vector laboratories, Burlingame, CA, USA) for 30 min, cells were incubated with primary antibodies against: a-SMA (Abcam, Cambridge, UK), prolyl 4-hydroxylase (Acris antibodies, Hiddenhausen, Germany), Phospho-SR proteins (Zymed Laboratories, San Francisco, CA, USA), and SRp20 (Zymed Laboratories, San Francisco, CA, USA) and with secondary antibodies: Alexafluor 555-conjugated goat anti-mouse antibody and Alexafluor 555-conjugated goat anti-rabbit antibody (both from Molecular Probes Invitrogen, Eugene, OR, USA). To stain nuclei, cells were incubated with DAPI (Molecular Probes Invitrogen, Eugene, OR, USA). Glasses were mounted with mounting media (Dako, Glostrup, Denmark) and photographed using a TE2000-E fluorescence microscope (Nikon, Tokyo, Japan) equipped with a DXM1200C camera (Nikon).
RNA extraction and cDNA synthesis
Cells were grown under standardized conditions with or without TGF-β1 (10 ng/mL) for 6, 24, or 48 h. Total RNA was isolated from cells using RNeasy (Qiagen, GmBH, Hilden, Germany) according to the manufacturer´s instructions. The quantity of RNA was measured by spectrophotometry using a NanoDrop ND-100 (Nano Drop Technologies, Delaware, MD, USA). Total RNA (1 μg) was reverse-transcribed using superscript II according to the manufacturer’s manual (Invitrogen, Carlsbad, CA, USA) and stored at −70°C.
Five μL of cDNA (diluted 1:250) was mixed with 15 μL SYBR-green mixture (PE Biosystems, Foster City, CA, USA) and amplified by real-time RT-PCR using Stratagene MX3005P QPCR system (Agilent Technologies, Santa Clara, CA, USA). Initially the samples were held for 2 min at 50°C then for 10 min at 95°C; they were then cycled for 40 cycles of 30 s at 95°C, 1 min at 58°C, and 1 min at 72°C. Each sample was analyzed in triplicate. Reactions were performed using MX3000P 96-well plates (Agilent Technologies, Santa Clara, CA, USA). All primers were constructed using the online Primer 3 program (http://frodo.wi.mit.edu/primer3/) and they were ordered from A/S DNA Technology (Risskov, Denmark). The following primers were used. Fibronectin forward: CGA TCA CTG GCT TCC AAG TT, and reverse: TCC GAG CAT TGT CAT TCA AG. Fibronectin-EDA forward: AAT CCA AGC GGA GAG AGT CA, and reverse: CGT AAA GGG CTC AGC TCA AG. S18 forward: CGA ACG TCT GCC CTA TCA AC, and reverse: TGC CTT CCT TGG ATG TGG TA. All primers were tested for specificity by sequence alignment in the PubMed nucleic acid database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Data are based on three individual sets of experiments.
Purification of nuclei
The nuclei were purified according from a protocol previously described . Briefly, the cells were harvested in 60 mM KCl, 15 mM NaCl, 0.15 mM Spermine, 0.5 mM Spermidine, 15 mM Hepes, and 14 mM Mercaptoethanol supplemented with 0.2% (v/v) Nonidet p40, aprotinin (1 μg/mL), leupeptin (1 μg/mL), pepstatin A (1 μg/mL), PMSF (10 μg/mL) and 0.3 M sucrose. The cell suspension was then homogenized with 60 strokes at 2,000 rpm (Labortechnik, Berlin, Germany) and the homogenate was filtered with 100 μm filter paper (Schleicher&Schuell, Dassel, Germany). The nuclei were counted in a Bürker chamber and the purity was assessed and protein content was determined by Bradford protein reagent kit (Pierce, Rockford, IL, USA).
Labeling with the acid-cleavable ICATTM reagent and isolation of cysteine containing peptides
Fibroblast nuclei were lysed in a buffer containing 6 M Guanidium-HCl (pH 8.5), 1% Triton X-100, and 50 mM Tris HCl, followed by sonication. Starting with 500 μg for control and TGF-β1 stimulated, each sample was reduced by adding 10 μl of 50 mM TCEP (Tris(2-carboxyethyl)phosphine) and boiled for 10 min. The samples were allowed to cool and the acid cleavable ICATTM reagent (all the materials in this section are from the ICATTM reagent kit Applied Biosystems, Framingham, MA, USA) was added: the light reagent to the control and heavy reagent containing nine 13 C to the TGF-β1-treated sample. Alkylation was allowed to complete for 2 h, at 37°C. The two samples are combined and acetone precipitated in order to remove the guanidium-HCl and the unreacted ICATTM reagent. The pellet was dissolved in a buffer consisting of 50 mM Tris (pH 8.5), 5 mM CaCl2, and 10% acetonitrile. Trypsin was added at a 1:40 enzyme/substrate ratio in two additions once at the start of the digestion and once more, 2 h later. Digestion was completed at 37°C overnight.
Following digestion the sample was diluted into 25% acetonitrile, pH 3.0 in order to reduce the buffer concentration below 10 mM. The resulting peptide mixture was injected to a PolyLC (4.6 × 100 mm Polysulfoethyl A) SCX column on Vision workstation (Applied Biosystems, Framingham, MA, USA) at a flow rate of 1 mL/min using a binding buffer (buffer A) 10 mM KH2PO4, 25% acetonitrile, pH 3 and an elution buffer (buffer B) of 350 mM KCL, 10 mM KH2PO4, 25% acetonitrile, pH 3. The gradient was 0% B to 10% B in 2 min, 20% B in 15 min, 45% B in 3 min, 100% B in 10 min, and an additional 8 min hold at 100% B. Twenty-three SCX fractions of 1.5 mL volume were collected.
The pH was adjusted for each SCX fraction to 7.2 with 10× PBS buffer (pH 10) and injected to the avidin affinity column as prescribed by the manufacturer (Applied Biosystems, Framingham, MA, USA). Cysteine-containing peptides bound to the avidin column were washed three times: 1 mL of Wash 1, 1 mL of Wash 2, and 1 mL of de-ionized water. Elution of the cysteine-containing peptides was performed with 800 μL of avidin elution buffer. The samples were then concentrated by a speed-vac to dry, cleaved with the ICATtm cleaving reagent containing 95% TFA and concentrated again to remove the cleaving reagent. The samples were then taken up in 2% acetonitrile, 0.1% TFA for re-injection for a nanoHPLC MS and MS/MS analysis.
Protein identification and quantification by LC-MS and -MS/MS
Avidin purified SCX fractions were subjected to MALDI and ESI HPLC-MS and MS/MS analysis in a 50:50 split. For the MALDI based workflow, these fractions were injected in the HPLC loading buffer onto a 100 μm × 15 mm C18 Magic column (Auburn, Michrom Bioresources, CA, USA) using a CapTrap pre-column (Auburn, Michrom Bioresources, CA, USA). Mobile phase A was 2% acetonitrile, 0.1% TFA, mobile phase B was 85% acetonitrile, 5% n-PrOH, 10% water, 0.1% TFA. HPLC elution was carried out at a 1-μL/min flow rate on an Ultimate nanoHPLC workstation (Dionex-LC Packings, Hercules, CA, USA). The HPLC elution from the column was collected at 20-s intervals on the MALDI plate using a Probot fraction collector (Dionex-LC Packings). Forty-eight-min HPLC elution was collected to the MALDI plates as an array of 12 × 12 sample spots. The HPLC eluent was mixed with the MALDI matrix (7.5 mg/mL α-cyano-4-hydroxycinnamic acid dissolved in 60:40 acetonitrile-water containing 0.15 mg/mL dibasic ammonium-citrate) through a mixing tee (Upchurch, WA, USA) at a flow rate of 2-μL/min. The most abundant, middle-fractions of the SCX separation were spotted onto two plates using a 90-min HPLC gradient.
MALDI plates were analyzed in automated mode on the AB4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA, USA). First the MS spectra were collected from the entire HPLC run. Then, using an in-house developed program, MS/MS precursors were selected by applying an exclusion algorithm to eliminate: (a) redundant precursors carrying over multiple HPLC fractions; and (b) using only the more abundant members of peptide pairs for MS/MS analysis. MS/MS spectra were acquired using up to 2,500 laser shots/precursor unless the predefined signal-to-noise level in the MS/MS acquisitions was achieved sooner. The MS/MS data were submitted for database searching as a batch to Mascot (http://www.matrixscience.com) through its automation interface of Mascot (Mascot Daemon). The non-redundant NCBI protein database was used. A detailed description of the acceptance criteria for the database searching results will be described elsewhere. In brief, tryptic peptides containing arginine residues were accepted at a Mascot score >20, peptides not containing arginine at a Mascot score >25. Peptides with less-than-significant Mascot score were thoroughly inspected, considering the correlation of peptide basicity with SCX fraction numbers, presence of characteristic high-energy CID fragments, and accurate mass measurements through internal calibration of the MS spectra using the masses of confidently identified peptides as internal mass references.
ESI based LC-MS/MS analyses were carried out using an Ultimate nanoHPLC system (Dionex-LC Packings, CA, USA) on a 75 μM × 150 mm Picofrit C18 column at a 300-nl/min flow rate with a gradient of 5% B to 30% B over 60 min. Mobile phase A was 2% acetonitrile, 0.1% formic acid, mobile phase was 85% acetonitrile, 5% n-PrOH, 10% water, 0.1% formic. A CapTrap (Michrom Bioresources, CA, USA) precolumn was used to preconcentrate the sample.
A platinum electrode was placed behind the HPLC column using a microtee (Upchurch, WA, USA) in order to apply the electrospray voltage (3 kV) for LC-MS/MS analysis. LC-MS/MS was performed using a QSTAR quadrupole time-of-flight instrument (Applied Biosystems, CA, USA). The instrument was set up to perform a 1-s MS scan (300–1500 Da) followed by three most abundant components (two to four charges were allowed) exceeding an intensity threshold of 35 counts. To improve the protein coverage the samples were halved and the analysis was replicated. LC-MS and MS/MS analyses were processed by the ProICAT software (Applied Biosystems, Foster City, CA, USA). Database search results were accepted at >95% confidence interval. Database searching and quantification results from multiple MS platforms were consolidated by parsing all the qualitative and quantitative peptide results into an Oracle database. The set of all the identified peptides were assigned to a minimum set of proteins that could explain all the confidently identified peptide sequences. The abundance ratios of peptide pairs labeled with the heavy (9 × 13 C) and light (0 × 13 C) ICATTM reagents were normalized to the median of all peptide ratios in order to generate normalized expression ratios. Expression ratios of peptides matching to the same protein were averaged to generate expression ratios at the protein level.
Data are expressed as mean ± SEM. Student’s t-test was used to evaluate the differences of the means between groups. Differences were considered significant at P < 0.05. All analyses were performed using GraphPad Prism software version 4.00 (GraphPad Software, San Diego, CA, USA).