Journal: Cell Death & Disease
Article Title: Downregulation of HS6ST2 by miR-23b-3p enhances matrix degradation through p38 MAPK pathway in osteoarthritis
Figure Lengend Snippet: MiR-23b-3p could enhance matrix degradation by means of regulating activity of p38 MAPK in human chondrocytes under TNF-α treatment. a , b SW1353 cells were transfected by mimic miR-23b-3p ( a ) or anti-miR-23b-3p sequence ( b ) with or without TNF-α for 24 h, and the protein expression of phosphorylation form of p38 MAPK (p-p38), total p38 MAPK, and MMP13 was detected by western blotting. Asterisk (*): compared with the mimic NC ( a ) or anti-NC group ( b ). c SW1353 cells were treated with mimic miR-23b-3p with or without p38 MAPK inhibitor SB203580 (10 μM) under stimulation of TNF-α. The protein expression of MMP13 was determined by western blotting. Asterisk (*): compared with the mimic NC group. d Interfering efficiency of siRNAs against p38 MAPK was determined by western blotting under 50 nM p38 siRNA, 50 nM negative control (NC), and Mock (transfection regent only) transfection for 48 h. e Under stimulation with TNF-α, SW1353 cells were transfected by mimic miR-23b-3p under treatment with si- p38 mixture (containing three siRNA target sequences), and p-p38, total p38, and MMP13 were determined by western blotting. Asterisk (*): compared with mimic NC or si-NC. Each relative expression of phosphorylation form was normalized by the total form. GAPDH was used as internal controls in western blotting detection. Mann–Whitney U test was used to identify statistical differences between two groups. Asterisk (*) or hash ( # ) stands for P value
Article Snippet: SW1353 cells were seeded in 24-well plates at 3 × 104 for 24 h. After transfection of miRNAs or plasmids, the chondrocytes were incubated with TNF-α (10 ng/ml, Sino Biological lnc, catalog HG10602-M) for another 24 h. In order to determine the matrix content, the chondrocytes were fixed in 4% buffered paraformaldehyde for at least 20 min and stained with 1% toluidine blue for 10 min (miRNA or siRNA transfection) or 15 min (plasmid transfection).
Techniques: Activity Assay, Transfection, Sequencing, Expressing, Western Blot, Negative Control, MANN-WHITNEY