Human recombinant visfatin

Human OA chondrocytes, at the first passage, were plated in 6-well dishes at a starting density of 1 × 10⁵ cells/well until they became confluent. Human recombinant visfatin (Sigma–Aldrich, Milan, Italy) was first dissolved in phosphate buffered saline (PBS) (Euroclone, Milan, Italy), according to the manufacturer’s instructions and then it was diluted in the culture medium immediately before the treatment to reach the final concentration required.
The cells were cultured for 24 h in DMEM with 0.5% FBS and visfatin at concentration of 5 μg/mL and 10 μg/mL. The concentrations of the adipokine used in this in vitro study were selected according to those used by other authors [13 (link),18 (link)]; the final concentrations were chosen based on the best results obtained in terms of viability.
Afterwards, cells were pre-incubated for 2 h with 1 μM BAY 11-7082 (NF-κB inhibitor, IKKα/β, Sigma–Aldrich, Milan, Italy) and then treated 24 h with the tested concentrations of visfatin.
After the treatment, the media were removed, centrifuged and stored at −80 °C, while the cells were immediately processed to carry out flow cytometry analysis and quantitative real-time PCR.

T/C-28a2 cell line and human OA chondrocytes, at the first passage, were plated in 6-well dishes at a starting density of 6 × 10⁶ cells/well until they became confluent. Human recombinant visfatin (Sigma–Aldrich, Milan, Italy) and human recombinant resistin (BioVendor, Rome, Italy) were first dissolved in phosphate buffered saline (PBS) (Euroclone, Milan, Italy), according to the manufacturer’s instructions, and then they were diluted in the culture medium immediately before the treatment to reach the final concentration required.
The cells were treated for 24 h with visfatin at concentration of 5 μg/mL and 10 μg/mL or resistin 50 ng/mL and 100 ng/mL. The concentrations of the adipokines used in our in vitro study were selected according to those used by other authors [12 (link),39 (link)]; the final concentrations were chosen based on the best results obtained in terms of viability.
After the treatment, the media were removed, centrifuged, and stored at −80 °C; the cells were immediately processed to carry out cell viability assay, flow cytometry analysis, and quantitative real-time PCR.
Afterwards, the cells were pre-incubated for 2 h with 1 μM BAY 11-7082 (NF-κB inhibitor, IKKα/β, Sigma–Aldrich, Milan, Italy) and then treated 24 h with the tested concentrations of visfatin and resistin. Subsequently, the gene expression of MMP-1, MMP-13, and Col2a1 was evaluated.

Human OA chondrocytes were plated in 6-well dishes at a starting density of 1 × 10⁵ cells/well until 85% confluence. Human recombinant visfatin (Sigma–Aldrich, Milan, Italy) was dissolved in phosphate buffered saline (PBS) (Euroclone, Milan, Italy), in accordance with the manufacturer’s instructions, and then directly diluted in the culture medium for the treatment in order to obtain the final concentration required.
The cells were cultured in DMEM enriched with 0.5% FBS and 2% P/S, and stimulated for 24 h with visfatin at concentration of 10 μg/mL, according to previous studies [27 (link),29 (link),47 (link)]. Some dishes were pre-incubated for 4 h with 10 μM of nicotinamide phosphoribosyltransferase inhibitor, FK866 (Sigma–Aldrich, Milan, Italy) [63 (link)].
After the treatment, the cells were recovered and immediately processed to carry out flow cytometry, quantitative real-time PCR, and western blot analysis.