Figure 4.

Chemical inhibition of EZH2 methyltransferase does not lead to an upregulation of early chondrocyte markers in CM+ectoderm. (A and E) Schematic demonstrating the isolation of primary CM+ectoderm fibroblasts. GSK126 is specific to EZH2, and UNC1999 is specific to both EZH2 and EZH1. GSK126 and UNC1999 inhibit EZH2’s methyltransferase activity. (B and F) Western blots demonstrating reduction/loss of H3K27me3 level following incubation with GSK126 ((IC50 = 75–100 nm) or UNC1999 (IC₅₀ < 10 nM for EZH2 and 45 nM for EZH1). (C, D, and G) qPCR analysis of the expression of Sox9 and Col2a1 following inhibition of EZH2. GSK126: n = 5 mutants and 6 controls for Sox9, and n = 3 mutants and controls for Col2a1. UNC1999: n = 7 mutants and 9 controls. CM, cranial mesenchyme; n.s., not significant; qPCR, quantitative polymerase chain reaction. * P < 0.05; ** P < 0.01.