Fig. 2

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