S1810 500

Immortalized osteoblasts from icap-1^−/−; β1 integrin flox/flox mice were generated as described previously (Bouvard et al., 2007 (link)). These cells were infected or not by adenoCre viruses from gene transfer vector core (University of Iowa) in order to obtain β1 integrin-null cells. The icap-1 null cells were incubated with retroviral particles to obtain rescued cells expressing ICAP-1 WT. The cells were selected with 1 mg/ml puromycin to produce cell populations with heterogeneous ICAP-1 expression levels. Cells were maintained in culture in DMEM (#31966-021; Life Technologies) supplemented with 10% FBS (#S1810-500; Dominique Dutscher), 100 U/ml penicillin, and 100 µg/ml streptomycin (#P06-07100; PAN Biotech) at 37°C in a 5% CO₂-humidified chamber. For all experiments, cells were washed with PBS (#L0615-500; Dominique Dutcher), detached using trypsin (#L0615-500; Dominique Dutcher), and treated with 1 mg/ml trypsin inhibitor (#T6522; Sigma-Aldrich). Cells were then plated in DMEM containing 10% FBS for 4 h and then the appropriate analysis was carried out. Where needed, a serum-free medium OptiMEM was used (#51985-026; Life Technologies) as substitute. The four osteoblast clones—icap-1^+/+; icap-1^−/−; β1 integrin^{floxed/floxed}; β1 integrin^{floxed/floxed}; and icap-1^−/−—were infected using lentiviral infection system from Invitrogen with pLenti–murine β3 integrin-GFP vector.