Cell culture and transient transfections

NIH-3T3, Swiss-3T3 fibroblasts, and paxillin-deficient murine fibroblasts were grown at 37 °C (10% CO₂) in DMEM with 4500 mg/L glucose (Sigma-Aldrich), supplemented with 10% heat inactivated FBS (PANBiotech), 1% penicillin-streptomycin (Gibco), and 2 mM glutamine (Sigma-Aldrich). Transient transfections were performed 24 h after seeding cells, with jetPRIME (Polyplus Transfection) according to the manufacturer’s recommendations.

Paxillin knock-out cells were generated by CRISPR-Cas9 technology. Three separate guide RNAs were used to target the mouse paxillin gene (Santa Cruz Biotechnology, sc-422546). Plasmids expressing gRNA together with Cas9 and GFP were transiently transfected into SV-40 immortalized pre-osteoblast cell line (αv^f/fβ1^f/f) as previously reported⁸¹. Single cells were FACSorted based on GFP expression and growing clones were further characterized for the loss of paxillin expression by western-blot and immunofluorescence.