Aspectos cuantitativos del Estudio I

To determine the protein expression of cellular and secreted fibulin-1 and CXCL1, human colon fibroblast and CRC cells were seeded in 6-well plates and treated with or without IL-6 as described above. Supernatant was collected and spun at 16,000g at 4C for 5 minutes to remove cellular debris. Total cellular protein was extracted by using lysis buffer containing 20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM Na2EDTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% sodium deoxycholate and cOmplete protease inhibitor cocktail, mini (Roche). Cell lysates were collected by scraping the cells off the plates with chilled lysis buffer and a cold plastic cell scraper (Greiner Bio-One), cell suspension was then constantly agitated at 4C for 30 minutes with a tube rotator before centrifuging at 16,000g at 4C for 20 minutes to pellet cellular debris. Cell lysate and supernatant samples were concentrated by freeze drying the samples overnight with a FreeZone -105°C 4.5L cascade bench top freeze dry system (Labconco). Freeze dried samples were reconstituted with 200 μL of MilliQ water.

Total protein was determined by the Pierce BCA protein assay (Thermo Scientific) according to the manufacture’s protocol. Briefly, protein concentration in cell lysate and supernatant samples was measured using a colourmetric protein assay based on a shift in absorbance of BCA upon binding to protein. Cell culture samples were diluted (1:10) with MilliQ water and 25 μL of this diluted sample was added to 200 μL BCA working reagent and incubated at room temperature for 30 minutes. Samples were quantitated in duplicate using a bovine serum

Chapter 3 In vitro investigation of fibulin-1 expression and functional role in colorectal cancer

Page | 114 albumin as a protein standard (0 – 2 mg/mL) and absorbance was measured with a FLUOstar Omega spectrophotometer (BMG Labtech) at 562 nm.

3.3.3 Western blotting

3.3.3.1 Preparation of methanol-free Coomassie staining protocol

Coomassie Brilliant Blue (G-250) (60 mg) was dissolved in 1 L distilled water by stirring for 2 – 4 hours over a magnetic hotplate before the addition of 6 mL of HCl and stored at room

temperature, covered.

3.3.3.2 Gel electrophoresis

Soluble and cellular fibulin-1 were detected by Western blotting (194) with 1.2 µg of cell lysate and 12 µg of supernatant loaded in each well and each gel loaded with a positive (1% FBS), negative (100 ng fibronectin) and normalising (1:500 healthy plasma sample) control. Samples were aliquoted into working loading volumes to ensure the number of freeze thaw cycles for the samples do not exceed three cycles. Proteins were separated on 10% SDS polyacrylamide gels at 125 V for 70 minutes, transferred to PVDF membranes at 25 V for 1.5 hours and

blocked with 5% skim milk/2% BSA/TBS/0.1% Tween-20 blocking solution at room temperature for 1 hour. All antibodies were diluted in 2% BSA/TBS/0.1% Tween-20. Membranes were incubated with mouse anti-human fibulin-1 monoclonal antibody (clone B-5) (Santa Cruz, 0.2 μg/mL for supernatant samples and 0.3 μg/mL for cell lysate samples) at 4C overnight. Following five 3 minute TBS/0.1% Tween-20 washes, membranes were then incubated with goat anti-mouse IgG-HRP polyclonal antibody (DAKO, 0.2 μg/mL for supernatant samples and 0.3 μg/mL for cell lysate samples) at room temperature for 1 hour. After washing, blots were visualised and exposed with Immobilon Western Chemiluminecent HRP substrate (Millipore) for 3 minutes, bands were analysed using ChemiDoc MP Imaging system (Bio-Rad), and the amount of protein detected in each sample was determine based on the densities obtained with Image Lab Version 4.0.1 (Bio-Rad).

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3.3.3.3 Re-probing membrane for GAPDH

Cell lysate PVDF membranes were washed with TBS/0.1% Tween-20 wash to remove the Immobilon Western Chemiluminecent HRP substrate (Millipore) prior to striping. The membrane was then incubated with Pierce Restore™ Western Blot Stripping Buffer (Thermo Scientific) for 15 minutes at room temperature and washed once again before blocking the membrane for an hour at room temperature as previously mentioned above. Sufficient removal of antibodies was tested with Immobilon Western Chemiluminecent HRP substrate (Millipore) before re-probing the membrane with mouse anti-human glyceraldehydes 3- phosphate dehydrogenase (GAPDH) monoclonal antibody (Millipore, 0.067 μg/mL) at 4C overnight. Following five 3 minute TBS/0.1% Tween-20 washes, membranes were incubated with goat anti-mouse IgG-HRP polyclonal antibody (DAKO, 0.013 μg/mL) at room temperature for 1 hour, washed, visualised, exposed for 30 secs and quantified as above.

3.3.3.4 Coomassie staining for supernatant samples

After protein transfer of supernatant-loaded samples, gels were washed with distilled water for 10 mins at room temperature twice. Coomassie stain was then added to the gel to stain overnight at room temperature, covered until staining has been achieved. Destaining was achieved with distilled water and gels were washed until sufficient destaining was achieved.

3.3.3.5 Analysis

The densities of fibulin-1 bands for each membrane were normalised according to the normalising factor of each membrane. For each membrane, the normalisation factor was calculated as the ratio of the 100kDa fibulin-1 band density detected in the normalising healthy plasma sample on the membrane against a representative membrane. Cellular fibulin- 1 was then normalised to GAPDH whilst secreted fibulin-1 was normalised to the 70kDa band in the Coomassie Blue-stained gel of each corresponding sample, and results were expressed as a fold change.

Chapter 3 In vitro investigation of fibulin-1 expression and functional role in colorectal cancer

Page | 116 Normalisa on of bulin 1 bulin 1 band density Normalisa on factor

GAPDH/Coomassie Blue band density