Alcian blue

Lipopolysaccharide (E. coli 055: B5), SDS, EDTA, hydroxylamine hydrochloride, thiobarbituric acid, RNA later, Bradford reagent, and protease inhibitor cocktail were procured from Sigma-Aldrich (St. Louis, MO, United States). TRIzol was purchased from Invitrogen, California, United States. de Man, Rogosa, and Sharpe (MRS) agar, glass slides, paraffin, hematoxylin, eosin, alcian blue, glycerol, hydrogen peroxide (H₂O₂), sodium bicarbonate (NaHCO₃), sodium carbonate (Na₂CO₃), sulfosalicylic acid, formalin, potassium chloride (KCl), and other chemicals were from HiMedia Laboratories, Mumbai, India; mouse-specific lipocalin-2, IL-6, IL-1β, and TNF-α ELISA kits were procured from Ray Biotech, Norcross, United States. SYBR-Green master mix and cDNA synthesis kits were purchased from Bio-Rad, United States. HPLC with a diode array detector was from Agilent Technologies, Singapore. NucleoSpin DNA Stool kits were procured from Macherey-Nagel, Düren, Germany. Antibodies (occludin, zona occludin, claudin, GAPDH, Muc-4, and Alexa Fluor 488) from Cell Signaling Technology (Danvers, MA, United States). HRP (horseradish peroxidase)-conjugated secondary antibody and DNase treatment AM1906 were purchased from Thermo Fisher, Leica CTR6, and Leica Biosystems, Germany. A confocal microscope was purchased from Carl Zeiss LSM-880, Germany.

The cultured cells were differentiated into osteogenic, adipogenic and chondrogenic lineage by culturing in osteogenic medium [DMEM supplemented with 10^− 8 M dexamethasone (Sigma, D4902), 10 mM β glycerophosphate (Sigma, G9422), and 50 μg/ml ascorbic acid], adipogenic medium [DMEM supplemented with 10 mM 3 isobutyl-1-methylxanthine (Sigma, 17018), 0.1 mM indomethacin (Sigma, 17378), 10 μg/ml insulin (Sigma, I6634), 10^− 6 dexamethasone] and chondrogenic medium (Stempro, Invitrogen) and confirmed by staining with Alizarin red (Sigma, A5533), Oil red O (Sigma, O0625) and Alcian blue (Himedia Laboratories, Mumbai, India: Cat no RM471-1 g staining, respectively).