Cabozantinib

To generate resistant cell lines, SNU449, SNU449-Axl^-, HLF and Huh7 were initially treated with 50% of the IC₅₀ concentration of Sora (TargetMol Chemicals, Boston, USA), Rego (TargetMol Chemicals, Boston, USA), and Cabozantinib (Cabo; TargetMol Chemicals, Boston, USA). The concentrations of TKIs were increased by 50% of the IC₅₀ every two weeks until 2 x IC₅₀ was reached and cells proliferated under high concentrations.

SW1353 chondrocytes were obtained from ATCC (USA) and cultured in F-12 medium supplemented with 10% FBS and 1% penicillin-streptomycin antibiotic solution. Cells were cultured in the T75 cell culture flask, the medium was changed 2-3 days. For experiments, cells were used at passages 3-8 and they were stimulated by 100 μg/ml AGEs to establish the in vitro OA model. To construct the SOX-9 knockdown SW1353 chondrocytes, cells were transfected with the siRNA targeting SOX-9 (siR-SOX-9) for 48 h, followed by determining the efficacy using the Western blotting assay and then treated with AGEs in presence or absence of Cabozantinib (Cat#T2586, TargetMol, USA).

The drugs used on cell cultures were: cabozantinib (3 and 5 µM; TargetMol), the small molecule MYC inhibitor 10058-F4 (20, 40, and 60 µM; TargetMol), and Omomyc (10 and 30 µM; kindly provided by L. Soucek and J. Whitfield, Vall d’Hebron Instituto de Oncología and Peptomyc) [48 ], currently in phase 1/2 clinical trial for other solid cancers (ClinicalTrials.gov Identifier: NCT04808362). Cell viability assays were performed as previously reported [29 (link)]. Briefly, cells were seeded in 150 µL volume of medium per well in 96 well plates (10,000 or 3000 cells/well for 48 h or 72 h treatment, respectively) in the presence of 10 % serum. After 24 h, inhibitors were applied, either individually or in combination, at the indicated concentrations. After 48 h and 72 h, cell viability was assessed with the Cell Counting Kit-8 colourimetric assay (TargetMol). Colourimetric signals were measured with a luminometer microplate reader (Berthold). Cell viability was normalised to non-treated (NT) cells. Data obtained from viability assays are the mean of three to six independent experiments performed in triplicate. To classify the effects of combined treatment in synergistic, additive, or antagonistic, the Bliss independence method was applied [59 (link)].