Fibronectin

Collagen (Coll, purified bovine Type I collagen, CBPE2, Symatese S.A.S) and Fibronectin (Fn) (human plasma Fibronectin, 11051407, Roche Diagnostics Pty Ltd.) were used in this study as a single and double-layered coating. Fibronectin was used at a concentration of 2 μg/mL in PBS (120 ng/cm²), Coll was used at 1 mg/mL dissolved in distilled water (60 μg/cm²) as per manufacturer’s guidelines. A collagen coating at this concentration has been shown to enable fibril formation in vitro (Kreger et al., 2010 (link)).
The polymer samples were passively coated following the protocol previously described by Sgarioto et al. (2012 (link)) The Fn solution was allowed to adsorb on the TCPS for 45 min, at 37°C and washed with PBS 3 times for 10 min. The Coll was left to adsorb for 10 min at RT and then washed twice in PBS for 10 min. The collagen + Fibronectin (Coll + Fn) coating was done similarly except, Coll was coated first followed by the Fn.

Gelatin (denatured collagen I, from bovine skin), vitronectin (from human plasma), bovine serum albumin (BSA, fraction V) and Efavirenz were obtained from Sigma-Aldrich (Milan, Italy). Human recombinant IL-1β, TNF-α, and IFN-γ, and fibronectin from human plasma were purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA). MAbs directed against the α5β1, αvβ3, αvβ5, or α6β4 integrins were obtained from Chemicon-Merck-Millipore (Darmstadt, Germany). The anti-CD31 (PECAM-1) mAb was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The Tat peptides (11–25), (46–60), and (66–80) were from UFP Service, University of Ferrara, Italy. Phosphate-buffered saline (PBS) solution, cell growth medium (RPMI 1640) and media supplements were obtained from Invitrogen-Life Technologies (Milan, Italy). Fetal bovine serum (FBS) was from HyClone (Logan, UT, USA).

DECs, labelled with the fluorescent dye FAST DiI (Molecular Probes, Invitrogen), were seeded onto 8-chamber culture slides (BD Biosciences Discovery Labware, Milan, Italy), previously coated with 2 µg/cm² fibronectin (Roche). After fixation with 1% paraformaldehyde and permeabilization with FIX & PERM kit, solution B (Società Italiana Chimici, Rome, Italy), cells were incubated with 10 µg/mL primary monoclonal antibody (cloneF8/86) mouse anti-human vWF (Dako) or mouse anti-human vascular endothelial (VE)-cadherin (kindly provided by Prof. Dejana from the Institute of Molecular Oncology, Milan, Italy) for 1 h at room temperature (RT), followed by incubation with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Dako) for 1h at RT. Images were acquired with a Leica DM3000 microscope (Leica, Wetzlar, Germany) and collected using a Leica DFC320 digital camera (Leica).

Live cell microscopy was performed to compare migratory capacities of SN-stimulated vs. unstimulated C32 cells, using a Zeiss Axio Observer Z.1 microscope equipped with AxioVision 4.8 software (Carl Zeiss AG, Oberkochen, Germany). C32 cells were seeded on a glass coverslip coated precoated with 50 µg/ml fibronectin (Roche Diagnostics) and allowed to adhere for 3 h in HEPES buffer. 10⁻⁶ M human SN (PolyPeptide Laboratories, Strasbourg, France) was added immediately before recording was started. Images were captured at 120-sec intervals at 100-fold magnification. The distance covered by each cell within 3.5 h was determined by Time Lapse Analyzer v01_32 software (AG Bioinformatics and Systems Biology, Institute of Neural Information Processing, Institute of Virology, University of Ulm, Germany).

3T3-L1 fibroblasts were differentiated on fibronectin (Roche Applied Science, Almere, the Netherlands) coated 96 well plates and differentiated. Mature adipocytes were transduced with Lentivirus particles containing FoxO1-Clover. FoxO1 is a direct and specific target of Akt; FoxO1 rapidly translocate from the nucleus to the cytoplasm in response to Akt activation (29 (link)). The transduction was followed by a 24 hour incubation with 0 or 1 mmol/L Mg²⁺ of which the last 6 hours adipocytes where incubated without FBS. Medium was replaced 30 minutes prior to starting live imaging; containing 0 or 1 mmol/L Mg²⁺, FBS free, phenol-free, supplemented with 1 μg/ml Hoechst 33342 (Sigma-Aldrich). Live imaging was performed using a ZEISS Axio Observer light microscope. Adipocytes were imaged for 30 minutes with a photo interval per 30 seconds. The cells were stimulated using a final concentration of 10 nM human insulin (Sigma-Aldrich) which was added between 30-60 seconds after the start of visualization. The relative intensity of fluorescent units in the cytosol/nucleus was analyzed by the following formula:
All values are normalized by dividing by the first measurement (t=0). Data was fitted using the four-parameter sigmoid dose-response curve. The maximum top value of this function was used as the fluorescent unit’s cytosol/nucleus ratio.

For the transwell migration assay, a 24-well Boyden chamber (8.0-μm pore size Corning, NY, USA) with fibronectin (Roche, Indianapolis, IN, USA) was used. TPC-1 or K1 cells (5 × 10⁴) were placed in the top chamber in medium with 1% FBS, and the medium supplemented with 20% FBS was filled in the lower chamber and served as a chemoattractant. After incubation at 37°C with 5% CO₂ for 24 h, the cells on the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with 1% Crystal Violet. The number of cells was counted using five random fields (200×).
For cell invasion assays, a Matrigel-precoated Transwell chamber (Corning) was used instead, and the procedures were performed as previously described above. The number of cells was counted using five random fields (200×).