Murine laminin

To investigate the trilineage differentiation potential, ASCs-_EXP and ASCs-_SVF of abd adipose tissue and ASCs-_EXP of rb and sc adipose tissue were initially expanded to P5 or P6 and then induced to adipogenic, osteogenic and chondrogenic differentiation in two experimental runs (n = 6). In all experiments, negative controls (NC) were included. The medium was changed every two to three days. In the case of adipogenic and osteogenic differentiation, cell culture vessels were coated with 2 µg/cm² murine laminin (Sigma Aldrich, Taufkirchen, Germany) in order to avoid cell detachment as far as possible, which was observed in Trachsel et al. due to the absence of any coating [59 (link)]. To ensure reproducibility, experiments were carried out in duplicate.

Immuno Maxisorb plates with 96 wells (Nunc; Thermo Fisher Scientific, Rochester, NY, USA) were coated with 10 μg/mL of human collagen I, chicken collagen II, human collagen IV, bovine fibronectin, murine laminin or BSA (Sigma-Aldrich) in PBS at 4 °C overnight and then washed with PBS and blocked with PBS plus 1% BSA for 2 h. Binding of bacteria to laminin coated on plates was studied by using PagN-expressing E. coli. Bacteria grown and induced to make PagN were centrifuged, suspended in PBS to 10⁷ CFU in 100 µL and added to laminin coated wells. Bacteria harboring empty plasmid pRS1 were used as a control. After incubation for 1 h at 37 °C, unbound bacteria were removed by three washing cycles, anti-PagN antiserum (1:500) was added, followed by wash cycles and incubation with goat anti-rabbit HRP-conjugated antibody (1:2000). After three wash cycles, bound antibodies were detected by using the 1-Step Turbo TMB ELISA substrate (Thermo Fisher Scientific) followed by 2 M sulfuric acid and measuring the absorbance at 450 nm. For the binding inhibition assays, double dilutions of heparin or heparan sulfate (400–0.4 µg/mL) were incubated with bacteria for 1 h and the mixtures were added to the laminin-coated wells for further processing as described above.

Antibodies against total integrin β1 and activated integrin β1 (HUTS-21) were purchased from BD Biosciences (Lincoln Park, NJ). Integrin β1 blocking antibody (P4C10) was purchased from Millipore (Billerica, MA). Antibodies against C1GALT1, GAPDH, and focal adhesion kinase (FAK) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody against phospho (p)-FAK was purchased from Cell Signaling Technology Inc. (Beverly, MA). Antibody against actin was purchased from GeneTex Inc. (Irvine, CA). Vicia villosa agglutinin (VVA) and peanut agglutinin (PNA) lectins were purchased from Vector Laboratories (Burlingame, CA). Human collagen IV, human fibronectin, murine laminin, bovine serum albumin (BSA), and protein de-glycosylation kit were purchased from Sigma (St Louis, MO).