Investigating collagen secretion in HSP47 KO MEFs

2.6 Investigating collagen secretion in HSP47 KO MEFs 2.6.1 Assessment of soluble collagen secretion

To quantitatively compare the amount of soluble collagen secreted from WT and HSP47 KO FBs, the amount of soluble collagen in the culture medium was assessed using the soluble Sircol^TM assay kit. A schematic outline of the experiment is summarized in Figure 11:

Figure 11. The measurement of soluble collagen secretion in MEFs. WT and HSP47 KO MEFs were resuspended in low serum medium (containing; DMEM, 0.5% FCS; 1% P/S; 148 µg/ml L-Ascorbic Acid 2-phosphate magnesium salt [AA]) and seeded on 6 well plates (3 x 10⁵ cells/well; 2 ml/well). Cells were cultured for 72 hrs in at 37 °C, 5% CO₂. Conditioned medium and cells were thereafter subjected to 1) conditioned medium analysis using the Sircol^TM assay kit, and 2) cells were counted using the Cellavista image platform (SynenTec Bio Services GmbH), respectively.

The cell conditioned medium from each well was transferred into fresh 2 ml microtubes and pelleted at 300 x g for 5 mins at RT, to remove cellular debris.

Conditioned medium (1 ml) was then transferred into clean 1.5 ml microtubes and

2. Materials and Methods

total soluble collagen in the conditioned medium was measured using the Sircol™

assay kit (Biocolor), according to manufacturer‟s instructions. Low serum medium (1 ml) cultivated on plates in the absence of cells were used as normalization controls.

Conditioned medium samples were initially treated with 200 µl ice-cold Isolation and Concentration reagent (provided by the manufacturer‟s), mixed and incubated overnight at 4°C to concentrate the collagen within samples, prior to measurements.

Next, samples were centrifuged at 15,300 x g and supernatants were then carefully removed leaving a remaining 100 µl and the invisible pellet of hydrated transparent collagen at the bottom of the microtube. Rat tail collagen reference standards (0, 1.25, 2.5, 5, 10 µg; provided by the manufacturer) were then prepared in 100 µl sterile water. Sircol dye reagent (1 ml) containing Sirius red in picric acid was added to each standard, control and sample tubes. Microtubes were then mixed by inversion and placed on a mechanical rocking platform (BioMetra) for 30 mins at RT in order to form collagen-dye complexes which precipitate out collagen from the soluble unbound dye. Tubes were then centrifuged again at 15,300 x g for 10 mins at 4 °C, and carefully inverted to remove the excess unbound dye reagent. Provided ice-cold Acid-Salt wash (750 µl) was gently added to tubes and gently mixed in order to remove any unbound dye on the surface of pellets and on the inside surfaces of tubes.

Microtubes were again centrifuged at 15,300 x g for 10 mins at 4 °C and acid-wash reagent was gently drained out from the tubes as much as possible without disturbing the red/pink precipitated collagen-dye pellet. To release and remove collagen from the bound dye, 250 µl of provided ice-cold Alkali reagent was added to all tubes.

Microtubes were mixed until the bound dye dissolved (~5 mins), and were subsequently transferred into individual wells (200 µl) of a 96-microwell plate.

The absorbance of standards, controls and samples at 555 nm was detected with the Flexstation^® 3 Multi-mode microplate reader, against water (blank) and the collagen amount of all conditioned medium samples (in 2 ml) were calculated from standard curves. The measured value of collagen in control tubes (containing low serum medium only) was subtracted from the sample values.

2. Materials and Methods

(x2) in 1X PBS, permeabilized in blocking buffer solution (see appendix A2), and labelled with Hoechst. Thereafter, the total number of fluorescence-labelled cell nuclei in each well was assessed using a high-throughput automated imaging platform (Cellavista; SynenTec Bio Services GmbH). The total amount of soluble collagen normalized to cell number (xg/cell) was thus determined from these two experiments, as calculated below:

* Determined via Soluble Collagen Sircol™ assay kit.per cell (xg/cellaaaaaaaaaaaaaaaaaa) was thus ca

# Determined via Cell nuclei counting/Cellavista.

2.6.2 Deoxychelate (DOC)-separation ECM components

WT and HSP47KO MEFs were assessed for differences in ECM secretion. The protocol for extraction of the deoxycholate (DOC)-insoluble ECM in MEFs was modified from (Van Duyn Graham et al., 2010; Midwood et al., 2002). WT and HSP47 KO MEFs were cultured at high seeding density (1 x 10⁵ cells/well, on 24-well plates) for 7 days in MEF medium containing: 148 µg/ml L-Ascorbic Acid 2-phosphate magnesium salt. Culture medium was removed and cells were washed once in 1X PBS. Cells and ECM suspensions were harvested in 300 µl cold DOC buffer (4

% DOC in 20 mM Tris-HCl, pH 8.8 and Protease inhibitors [Roche]), scraped off and transferred into 1.5 ml microtubes. The extracts were homogenized (x5) with a 27¹/₂ gauge needle and then pelleted at 18,000 x g for 20 mins at 4 °C. The supernatants (DOC-soluble fraction) were carefully collected and transferred into fresh 1.5 ml microtubes. The pellets were washed again with 200 µl DOC buffer and centrifuged at 18,000 x g for 20 mins at 4 °C. The supernatant was transferred into the DOC-soluble fraction, containing cellular material and components not incorporated into the ECM.

The DOC-insoluble pellet fraction containing ECM components (Van Duyn Graham et al., 2010; Midwood et al., 2002) was solubilised in 200 µl DOC insoluble buffer (1% SDS, 25 mmol/l Tris-HCl, pH 8.0, and protease inhibitors). DOC-insoluble ECM fractions were incubated at 95 °C for 5 mins.

2. Materials and Methods

Prior to SDS-PAGE analysis, protein content was quantified with a standard Pierce™ bicinchoninic acid (BCA) protein assay (ThermoFisher Scientific), to standardize loading protein concentrations (Smith et al., 1985). Proteins were resolved on 10% SDS-polyacrylamide gels (as described in Section 2.3.1.4). A rat tail collagen sample was used as a comparative molecular weight reference for collagen type I. To visualize all proteins resolved on gels from DOC insoluble ECM fractioned samples, coomassie blue staining was used to stain SDS-PAGE gels:

Resolved SDS-PAGE gels were carefully removed from casting chambers and were gently washed (x3) in deionized water for 5 mins at RT. Gels were then incubated for 1 hr at RT in Coomassie Brilliant Blue solution (G-250). Next, staining solution was carefully removed and SDS-PAGE gels were washed once in distilled water for 5 mins to remove any unbound dye, and then rinsed overnight at RT. Images of coomassie stained gels were taken on the ChemDoc^TM MP Imaging System (BioRad).

2.6.3 Mass spectrometric analysis of DOC-insoluble ECM components

Mass spectrometric (MS) analyses were performed by the Proteomics Core Facility at the University Medical Center Göttingen (Dr. C. Lenz; Prof. H. Urlaub).

2.6.3.1 Sample preparation

DOC-insoluble ECM fractions from WT- and HSP47 KO MEFs (n=1 sample/group) were reconstituted in 1× NuPAGELDS Sample Buffer (Invitrogen) and separated on 4-12 % NuPAGE Novex Bis-Tris Minigels (Invitrogen). Gels were stained with Coomassie Blue for visualization purposes, and each lane was sliced into 23 equidistant segments, regardless of staining. After washing, gel slices were reduced with dithiothreitol (DTT), alkylated with 2-iodoacetamide and digested with trypsin overnight. The resulting peptide mixtures were then extracted, dried in a SpeedVac, reconstituted in 2 % acetonitrile/0.1 % formic acid/ (v:v) and prepared for Nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS) as described previously (Atanassov and Urlaub, 2013).

2. Materials and Methods

2.6.3.2 NanoLC-MS/MS analysis

For MS analysis, samples were enriched on a self-packed reversed phase-C18 precolumn (0.15 mm ID x 20 mm, Reprosil-Pur120 C18-AQ 5 µm, Dr. Maisch, Ammerbuch-Entringen, Germany) and separated on an analytical reversed phase-C18 column (0.075 mm ID x 200 mm, Reprosil-Pur 120 C18-AQ, 3 µm, Dr. Maisch) using a 30 min linear gradient of 5-35% acetonitrile/ 0.1% formic acid (v:v) at 300 nl min^-1). The eluent was analyzed on a Q Exactive hybrid quadrupole/orbitrap mass spectrometer (ThermoFisher Scientific, Dreieich, Germany) equipped with a FlexIon nanoSpray source and operated under Excalibur 2.4 software using a data-dependent acquisition method. Each experimental cycle was of the following form: one full MS scan across the 350-1600 mass-to-charge ratio (m/z) range was acquired at a resolution setting of 70,000 Full Width at Half Maximum (FWHM), and Automatic Gain Control (AGC) target of 1 x 10⁶ and a maximum fill time of 60 ms. Up to the 12 most abundant peptide precursors of charge states 2 to 5 above a 2 x 10⁴ intensity threshold were then sequentially isolated at 2.0 FWHM isolation width, fragmented with nitrogen at a normalized collision energy setting of 25 %, and the resulting product ion spectra recorded at a resolution setting of 17,500 FWHM, and AGC target of 2 x 10⁵ and a maximum fill time of 60 ms. Selected precursor m/z values were then excluded for the following 15 s. Two technical replicates per sample were acquired.

2.6.3.3 Data processing

Peaklists were extracted from the raw data using Raw2MSMS software v1.17 (Max Planck Institute for Biochemistry, Martinsried, Germany). Protein identification was achieved using MASCOT 2.4 software (Matrixscience, London, United Kingdom).

Proteins were identified against the UniProtKB mouse reference proteome v2014.12, along with a set of 51 contaminants commonly identified in our laboratory. The search was performed with trypsin as enzyme and iodoacetamide as cysteine blocking agent. Up to two missed tryptic cleavages and methionine oxidation as a variable modification were allowed for. Search tolerances were set to 10 ppm for the precursor mass and 0.05 Da for fragment masses, and ESI-QUAD-TOF specified as the instrument type.

2. Materials and Methods

Scaffold software version 4.4.1.1 (Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0 % probability by the Percolator algorithm. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Protein identifications were accepted if they could be established at greater than 99% by the Percolator algorithm and contained at least 2 identified peptides. Protein hits that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. Proteins were annotated with GO terms from NCBI downloaded Feb 23, 2015 (Ashburner et al, 2000). Relative quantification of proteins in the samples was achieved by Analysis of Variance (ANOVA) of normalized Spectral Counts using a Benjamini-Hochberg-corrected p value of 0.05 to judge significance. To allow for the calculation of low abundance protein ratios, a minimum value of 3 spectral counts was introduced where necessary.

2.7 Fibroblast-derived ECM characterization