Journal: Arthritis Research & Therapy

Article Title: H3K27me3 demethylases regulate in vitro chondrogenesis and chondrocyte activity in osteoarthritis

doi: 10.1186/s13075-016-1053-7

Figure Lengend Snippet: The impact of trimethylated histone 3 lysine 27 (H3K27me3) inhibition on extracellular matrix production and chondrogenic gene expression. a Addition of GSK-J4 to chondrogenic media of human mesenchymal stem cells (MSCs) caused a reduction in collagen production as measured by Nile Red incorporation relative to viable cells (day 7, n = 3 individuals, 3 technical replicates per condition). b Jumonji domain-containing 3 ( JMJD3 ) expression was increased during chondrogenic differentiation of MSCs to form cartilage discs. Assessment of gene expression at days 7 and 14 of chondrogenesis revealed that H3K27me3 inhibition through GSK-J4 addition to chondrogenic media resulted decreased expression of SRY (sex determining region Y)-box 9 ( SOX9 ), collagen type II, alpha 1 ( COL2A1 ), aggrecan ( ACAN ) and collagen type X, alpha 1 ( COL10A1 ) ( c , d , f , g ) and increased expression of collagen type I, alpha 1 ( COL1A1 ) (e) ( n = 3 individuals). h MSCs were pre-treated with small interfering RNA (siRNA) against JMJD3 , lysine (K)-specific demethylase 6A ( UTX ) or non-targeting siRNA control prior to chondrogenic induction in Transwell culture. RNA was extracted and cDNA synthesized at day 7 of chondrogenesis, and expression of SOX9 , ACAN COL2A1 , COL10A1 and COL1A1 was assessed by quantitative reverse transcription polymerase chain reaction ( n = 4 patients, n = 2 technical replicates per patient). Dashed line represents expression level following MSC treatment with non-targeting siRNA control. i Proteoglycan content at day 14 in cartilage discs generated in Transwell cultures was reduced following addition of GSK-J4 to chondrogenic medium ( n = 3 individuals). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Data are presented as individual data points for biological replicates showing mean ± 95 % CI. a and i t test, b – h analysis of variance with Dunnett’s correction for multiple comparisons

Article Snippet: For cartilage disc generation, MSCs were resuspended in chondrogenic medium and 100 μl of MSCs at 5 × 10 6 cells/ml were pipetted onto 6.5-mm, 0.8-μm pore polycarbonate Transwell filters (Corning Costar, Corning, NY, USA) in a 24-well plate, centrifuged (200 × g , 5 min) before addition of 500 μl of chondrogenic medium to the lower well.

Techniques: Inhibition, Gene Expression, Expressing, Small Interfering RNA, Control, Synthesized, Reverse Transcription, Polymerase Chain Reaction, Generated