Protein biochemical methods

3.5.1. Protein isolation, precipitation, and quantification

For detection of secreted proteins in 2-D culture, the supernatant per well was taken. In 3-D culture, the culture medium of 100-200 spheroids collected in one 96-well was used. In order to concentrate proteins, the solution was incubated on ice for 20 minutes with a final concentration of 0.14% (w/v) Triton X-100 and 15.5% (w/v) trichloric acid (TCA).

Chapter 6 Regulation of Collagen Type XVI during in vitro Adipogenesis

Centrifugation with 18,000 x g for 20 minutes at 4°C resulted in a protein pellet that was washed two times with pre-chilled acetone. After air-drying, the pellet was resuspended in an appropriate volume of sample buffer.

For detection of proteins within the cell, cells were washed with PBS, harvested in lysis buffer (3.5 mM SDS, 8.3 mM Tris ad 250 ml, pH 7.4, adding protease inhibitor (complete mini)) and sonicated with a digital sonifier (Branson Ultrasonic Corporation, Danburg, CT, USA). Protein content was determined using the Bradford method [46]. Measurements were done in three biological replicates; one replicate was derived from one well for 2-D cultures and a number of approximately 200 spheroids at day 0 and 3 and of 100 spheroids at day 6 and 10, respectively, for 3-D cultures.

3.5.2. SDS-PAGE according to Laemmli

Table 2: Buffer concentrations for SDS-PAGE according to Laemmli.

Solutions Concentration of ingredients Resolving gel buffer 1.5 M Tris/HCl, pH 8.8

0.4% (w/v) SDS

Stacking gel buffer 0.5 M Tris/HCl, pH 6.8 0.4% (w/v) SDS

Acrylamide stock solution 30% (w/v) bisacrylamide

TEMED 100% triethylmethylethylendiamine

APS 10% (w/v) ammonium persulfate in water

Sample loading buffer

0.1 M Tris/HCl, pH 6.8 1% (w/v) SDS

20% (v/v) glycerol

5% (v/v) ß-mercaptoethanol 0.01% (w/v) bromphenol blue

The discontinuous sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli [47] was used to discriminate heterogeneous protein samples comprising different protein fractions. After addition of sample loading buffer to the protein samples, they were denatured for 5 minutes at 95°C and added into a 4.5% to 15% gradient

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resolving gel with a 4.5% stacking gel. Collagen XVI was detected by Western blot of 20 µg total protein. Concentrations of the ingredients are listed in Table 2. The gel run was performed at a maximum current of 20 mA. A pre-stained protein marker was always co-separated to determine the molecular weight of the sample (Precision Plus™ Protein All Blue Standard, BioRad, München, Germany).

3.5.3. Western blot analysis

Table 3: Buffer concentrations for Western blot analysis.

Solutions Concentration of ingredients Blotting buffer 50 mM Tris

380 mM glycine 0.1% (w/v) SDS 10% (v/v) methanol

1x TBST 140 mM NaCl

15 mM Tris/HCl 5 mM EDTA

0.1% (v/v) Tween 20 in TBS

Blocking solution 5% (w/v) dry milk powder in 1xTBST

Proteins were electrotransferred to a nitrocellulose membrane (Protran, Schleicher & Schuell, Germany) with the BioRad Mini Trans Blot Electrophoretic Cell (BioRad, München, Germany) in a sandwich of three layers whatman paper (BioRad, München, Germany), nitrocellulose membrane, polyacrylamide gel and again three layers of whatman paper. Per cm² of nitrocellulose membrane 0.8 mA current was applied for 60 to 75 minutes according to the fragment size of the protein. Prior to transfer, whatman paper and nitrocellulose membrane were soaked in blotting buffer (Table 3). After blotting, transfer of proteins was confirmed by a reversible Ponceau red staining of the membrane with a 0.2% (w/v) Ponceau S solution in 3% (v/v) TCA. After removal of the Ponceau red staining with water, unspecific binding sites were blocked with blocking solution (Table 3) for 1.5 hours at room temperature. After a short washing step in Tris-buffered saline Tween 20 (TBST), the membrane was incubated at 4°C overnight with the polyclonal guinea pig-anti-colXVI antibody (1:1,000) in blocking solution. Unspecificly bound antibody was removed in three

Chapter 6 Regulation of Collagen Type XVI during in vitro Adipogenesis

washing steps for 20 minutes each in TBST. The secondary goat-anti-guinea pig peroxidase-conjugated antibody was diluted 1:30,000 in blocking solution and the membrane was incubated for 60 minutes at room temperature. Washing steps were repeated as described for the primary antibody. Protein bands were detected by incubating the membrane with Super Signal West Femto Trial ECL reagent (Thermo Scientific, Rockford, IL, USA) for 1 minute.

Wrapped in clear foil, the luminescent signal was detected by application of an x-ray film for 10 seconds to 5 minutes (Amersham, UK).

3.5.4. Immunofluorescence

Cells grown on coverslips (Sarstedt, Nümbrecht, Germany) or entire spheroids were washed once with PBS, fixed in 4% (w/v) paraformaldehyde (PFA) in PBS overnight and washed thoroughly in PBS. The spheroids were embedded in Tissue-Tek (Hartenstein Laborbedarf, Würzburg, Germany), snap frozen and finally cut into 12 µm-thick cryosections. Serial sections were prepared from all spheroids and sections from the center region were used for histological evaluation. After dissolution of Tissue-Tek in water, cryosections were fixed in ice-cold acetone for 10 minutes at 4°C. Further procedures were performed with both cells in monolayer and cryosections of spheroids. Unspecific binding sites were blocked with 5%

(v/v) normal goat serum, 1% (w/v) BSA and protease inhibitor (complete mini, 1:5) in PBS for 60 minutes at 37°C. Guinea pig-anti-colXVI primary antibody was diluted 1:100 in blocking buffer and incubated at 4°C overnight. After washing with PBS, the goat-anti-guinea pig Alexa Fluor® 555 antibody diluted in PBS at a final concentration of 5 µg/ml was applied and incubated at 37°C for 1 hour. Nuclei were counterstained with 300 nM DAPI for 10 minutes and the slides were washed again with PBS before they were mounted with fluorescence mounting medium. All histological incubation steps were performed in humid chambers. The stained structures were visualized with confocal laser scanning microscopy (C1 confocal microscope, C4 camera and software Nikon, Germany).

3.6. Statistics

For TG accumulation, DNA measurements and qRT-PCR data, results are presented as mean values ± standard deviation. Statistical analyses were determined using GraphPadPrism v.5 (GraphPad Software, La Jolla, CA, USA). Differences between two groups were analyzed for significance using the unpaired Student’s t-test. Differences between multiple groups were analyzed for significance using one-way analysis of variances (ANOVA) with subsequent

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multiple comparisons according to Tukey’s post hoc test. A value of p<0.05 was regarded as statistically significant. For microarray analysis, the mean value and the values of both replicates are depicted.

4. Results

4.1. Gene expression of collagen XVI(α1) during adipogenesis of 3T3-L1 in 2-D and