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Quantification of cell number was performed as described 31 (link). Cells were serum starved for 2-4 h prior to treatment with different factors. At different time points, cells were harvested by trypsinization and viable cells were counted using trypan blue staining and a Neubauer chamber. As additional approach, viable adherent cells were stained, as described 32 (link), with crystal violet (Sigma-Aldrich, Sant Louis, Missouri). The absorbance of each plate was read photometrically at 590 nm using a plate reader (Powerwave XS, Biotek). Percentage of remaining viable cells was calculated with respect to control (untreated) cells.
MTT assay was performed according to manufacturer´s recommendations. Briefly, 20 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole bromide (MTT) reagent (Promega) was added to a volume of 180 µl of culture medium, and the plate was incubated for 2 h at 37°C. MTT reduction for formazan was measured at 620 and 492 nm (wavelengths corresponding to reagent and product of the reduction reaction) in a plate reader (Powerwave XS, Biotek). Difference between the absorbance of the product and reagent for each well was calculated.

The amount of collagen in the prepared bone powder was determined using a hydroxyproline (HYP) assay with an aliquot of the acid hydrolysate as described by Reddy and Enwemeka [18 (link)]. In summary, aliquots of the standard HYP (2–20 µL) prepared from stock solution (1 mg/mL of HYP in water) and the bone powder hydrolysate (at different volumes; 2.5 to 10 µL) were mixed with water in a total volume of 50 µL. Next, 450 µL of chloramine-T was added, mixed gently, and incubated for 25 min at room temperature. Then, 500 µL of Ehrlich’s aldehyde reagent was added, and the sample mixed and incubated at 65 °C for 20 min for the chromophore reaction to develop. Finally, absorbance was read at 550 nm using a microplate spectrophotometer (BioTek PowerWave XS, Winooski, VT, USA). Collagen content was calculated on the dry weight of the bone assuming 14% HYP in type I collagen [19 (link)].

The secretion of IL-6, IL-10, and IL-8 cytokines in PBMC supernatant was determined in duplicates by a sandwich ELISA according to methods reported by Ciliberti et al. [20 (link),22 (link)]. Plates were coated overnight at 4 °C with the capture antibodies, represented by mouse monoclonal antibody (mAb) anti-sheep IL-6, (Clone 4B6, Biorad Ltd., Hercules, CA, USA), anti-bovine IL-10 (Clone CC318, Biorad Ltd., Hercules, CA, USA), and anti-bovine IFN-γ (Clone CC330, Biorad Ltd., Hercules, CA, USA). As detecting antibodies, the rabbit polyclonal anti-ovine IL-6, the biotinylated mouse anti-bovine IL-10 mAb (Clone CC320, Biorad Ltd., Hercules, CA, USA), and the biotinylated anti-bovine IFN-γ antibody (clone CC302, Biorad Ltd., Hercules, CA, USA) were added, respectively. All the bovine antibodies involved in the sandwich ELISA demonstrated cross reactivity with ovine species. In each assay a standard curve was built using scalar dilution of the recombinant ovine IL-6 protein (Cusabio Biotech Co., Wuhan, China), recombinant bovine IL-10 (Biorad Ltd., Hercules, CA, USA), and recombinant bovine IFN-γ (Kingfisher Biotech, St Paul, MN, USA). The reading was set at 450 nm (Power Wave XS, Biotek, Charlotte, VT, USA). Data were expressed in nanograms of IL-6, IL-10, and IL-8 per milliliter.

RAW 264.7 cells (3 × 10⁴ cells/well) were exposed to the indicated concentrations of compounds 1–22 for 1 h and then incubated for an additional 24 h with LPS (1 µg/mL). At the end of the incubation, each culture supernatant was blended with the Griess reagent to determine NO production by RAW 264.7 cells. Optical density at 540 nm of the mixture was determined using a spectrophotometer microplate (PowerWave XS; Bio-Tek Instruments, Winooski, VT, USA).

Concentrations of IL-1β and IL-18 in cell culture medium or rat synovial fluid were measured with rat IL-1β enzyme-linked immunosorbent assay (ELISA) Kit (MultiSciences Biotech Co., Ltd., Hangzhou, China; 70-EK301B/3) and rat IL-18 ELISA Kit (Sigma-Aldrich; RAB1147) according to the supplier's protocols. Absorbance was measured at 450 nm wavelength using a PowerWave XS (Biotek, Winooski, VT, United States).

Cell viability was measured via a commercial colorimetric assay based on the reduction of the yellow tetrazolium dye MTT (Sigma-Aldrich) to purple formazan by NAD(P)H-dependent oxido-reductases in living cells. Thus, MTT reduction is a reflection of the number of viable and proliferative cells. We assessed cytotoxicity by comparing melanoma (MM96L, A2058, HTT144, JA, SKMEL28, A02, C001, C002), melanocytes and NFF cells. Briefly, 2,500 or 5000 melanoma and 8,000 or 4,000 (NFF & melanocytes) cells/well were seeded in a 96-flat microtiter well plate for 24 h to allow cell adhesion. Gomesin peptides were then added and MTT reduction measured after 48 h from the absorbance at 540 nm measured on a microplate reader (BIOTEK PowerWave XS). 0.1% SDS was used as a positive control (100% toxicity). The concentration of gomesin peptide causing 50% inhibition (IC₅₀) was determined was determined using GraphPad Software, Inc (USA).