Bovine chondrocytes were isolated from metatarsal joints obtained from a local abattoir within 24 h from death. Experiments were performed when primary chondrocytes reached 80% confluence (passage 0, P0) as previously described18 (link). C3H/10T1/2 cells (ATCC) were cultured according to the manufacturer’s instructions.
Murine femoral heads and patellae were explanted from 1 month-old male 129/Sv mice and processed for gene expression analysis (see below).
If not differently indicated, cells were stimulated in complete medium (DMEM/F-12 1:1 plus GlutaMax, 10% FBS, 1 mM sodium pyruvate, and 2% antibiotic antimycotic solution ThermoFisher Scientific) supplemented with human recombinant WNT3A (100 ng/ml; R&D), DKK1 (100 ng/ml, R&D), IL1B (20 ng/ml; InvivoGen), KN93 or its inactive control KN92 (10 μM; EMD Millipore), Autocamtide-2 related inhibitory peptide II, AIP, 5 μM; Calbiochem) or respective vehicles as indicated.
Murine femoral heads and patellae were explanted from 1 month-old male 129/Sv mice and processed for gene expression analysis (see below).
If not differently indicated, cells were stimulated in complete medium (DMEM/F-12 1:1 plus GlutaMax, 10% FBS, 1 mM sodium pyruvate, and 2% antibiotic antimycotic solution ThermoFisher Scientific) supplemented with human recombinant WNT3A (100 ng/ml; R&D), DKK1 (100 ng/ml, R&D), IL1B (20 ng/ml; InvivoGen), KN93 or its inactive control KN92 (10 μM; EMD Millipore), Autocamtide-2 related inhibitory peptide II, AIP, 5 μM; Calbiochem) or respective vehicles as indicated.
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