Collagen g

All reagents were of analytical grade or of the highest grade available. Antibiotic mixture of penicillin/streptomycin (10,000 U/mL/10,000 mg/mL), fungizone (250 mg/mL), and heat-inactivated fetal bovine serum (FBS) were obtained from GIBCO Invitrogen (Barcelona, Spain). Collagen G was obtained from Merck (Darmstadt, Germany). 4-Fluorobenzaldehyde (≥98%), collagenase from Clostridium histolyticum Type IA, desmosterol (≥84%), dexamethasone, ethylene glycol-bis-(2-aminoethylether)-N, N, N’, N’-tetraacetic acid (EGTA), gentamicin, insulin solution from bovine pancreas (10 mg/mL), methoxyamine hydrochloride (≥98%), N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane (BSTFA + 1% TMCS), O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA, ≥99%), sodium chloride (NaCl, ≥99.5%), thiazolyl blue tetrazolium bromide (MTT, ≥98%), thymol (≥98.5%), Triton X-100, trypan blue solution, Williams’ E medium, and all standards used throughout the work were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Methanol (≥99.9%) and pyridine (≥99%) were purchased from VWR (Leuven, Belgium).

HEK293T cells were purchased from ATCC (CRL-3216). For the cultivation of HEK293T cells, Dulbecco’s modified Eagle medium (DMEM, Pan Biotech GmbH, Aidenbach, Germany) supplemented with 10% FCS (Pan Biotech GmbH), 100 U/mL penicillin, and 100 µg/mL streptomycin (Pan Biotech GmbH) was used. The cells were cultured at 37 °C and 5% CO₂ in constant humidity. Before cell seeding, culture flasks and multiwell plates were coated with collagen G (0.001% in PBS, Merck, Darmstadt, Germany).

To investigate the interaction of control and 72 h SHI-treated cells with extracellular matrix proteins (ECM), 24-well tissue culture plates were pre-coated with collagen G (200 µg/mL; Merck, Darmstadt, Germany), fibronectin (10 µg/mL; Gibco™, Thermo Fisher Scientific, Darmstadt, Germany) or laminin (10 µg/mL; Corning GmbH, Kaiserslautern, Germany) overnight at 4 °C. Wells covered with PBS served as a background control for the unspecific binding. To minimize non-specific cell adhesion, plates were blocked 1 h before assay with 1% BSA.
To determine the interaction of tumor cells with the vascular endothelium, 1.25 × 10⁵ HUVECs were plated into 24-well-plates 16 h prior to an adhesion assay. For adhesion assays 1.25 × 10⁵ parental and sunitinib-resistant RCC cells, pre-stained with CellTracker Green CMFDA Dye (Invitrogen™, Thermo Fisher Scientific, Darmstadt, Germany) and pre-treated with SHI or diluent, were used per well and incubated for 1 h (adhesion to ECM) or 2 h (adhesion to HUVEC) at 37 °C in a humidified CO₂ incubator. Non-attached tumor cells were washed off with PBS, containing Mg²⁺ and Ca²⁺, and fixed with 2% glutaraldehyde. To score adhesion, the mean fluorescent intensity of attached cells was determined using a Sapphire Imager (Azure Biosystems, Munich, Germany). Values were presented as a percentage compared to untreated controls, set to 100%.