Bovine purecol

48-well plates were coated with human plasma fibronectin (2 μg/mL: Sigma-Aldrich, Cat# F0895) or bovine collagen type I (0,5 μg/mL: Bovine PureCol^®, Advanced BioMatrix, Carlsbad, CA, USA, Cat# 5005) and incubated for 2 h at 37 °C. After washing the coated plates twice with PBS, they were blocked with 2% BSA for 1 h at 37 °C, and the cells were washed twice with DMEM without FBS. 1 × 10⁵ cells/well were incubated for 45 min at 37 °C with clone 11,711 (10 μg/mL, mouse IgG1 isotype control, R&D Systems, MN, USA, Cat# MAB002), mAb 203E1 (10 μg/mL, integrin α11 antibody), P1E6 (5 μg/mL, integrin α2 antibody, Merck Millipore, Cat# MAB1950Z), and mAb 13 (5 μg/mL, integrin β1 antibody, BD Biosciences, San Jose, CA, USA, Cat# 552828). Following incubation, the non-adherent cells were carefully removed by washing twice with PBS containing Ca²⁺ (1 mM) and Mg²⁺ (0.5 mM). The cells were fixed with absolute ethanol for 10 min at room temperature, washed twice with distilled water, and stained with 0.1% crystal violet for 25 min at room temperature. The plates were washed three times with distilled water and the cells were lysed with 1% Triton X-100/PBS for 15 min. The lysates were transferred to a 96-well plate and the absorbance was read at 595 nm (Spectramax^® Plus 384, Molecular Devices, San Jose, CA, USA).

L230 enzymatic activity was measured as recently described^{22 (link)}. In brief, the assay was performed in reaction buffer (50 mM HEPES buffer pH 7.4, 150 mM NaCl) at 37 °C for 1 h with 1 μM L230 enzyme, 10 μM FeSO4, 100 μM 2-OG, 500 μM ascorbate, 1 mM dithiothreitol, 0.01% Triton X-100, and 1 mM viral collagen peptide substrate ETGLKGII or 4 μM bovine skin collagen substrate containing no telopeptides (Bovine PureCol^®, Advanced BioMatrix). With the exception of L230 recombinant protein and bovine skin collagen, all reagents were prepared immediately before use. All of these reagents were dissolved in reaction buffer with the exception of FeSO₄ and collagen, which was prepared in 10 mM HCl, and the pH of the reaction mixture was checked with pH papers to ensure that HCl did not change the overall sample pH. Bovine skin collagen was denatured by heating at 95 °C for 5 min and then chilled immediately on ice before use. L230 activity was measured by detecting succinate production with an adenosine triphosphate-based luciferase assay (Succinate-Glo™ JmjC Demethylase/Hydroxylase Assay, Promega, Madison, WI) according to the manufacturers’ instructions. Results shown are the mean values of triplicate biological samples in a single experiment. Each experiment was repeated once.