EZH2 directly regulates Mmp9 expression. (A) Mmp9 mRNA expression level in E12.5 FL cells (2 independent experiments). (B) Mmp9 protein level in coculture supernatant in panel G quantified by ELISA. (C) Mmp9 mRNA expression level in DMSO and 10 μM GSK126 (Ezh2 inhibitor) treated C166 cells (1 experiment). (D) ChIP-qPCR for Ezh2, H3K27me3, and H3K27ac at Mmp9 locus in DMSO or GSK126 treated C166 cells (3 independent experiments). (E) Representative immunofluorescence staining (2 independent experiments) of E12.5 FL endothelial cells collected post in vitro treatment with DMSO or 10 nM Mmp9 inhibitor using antibodies against CD31 (red, endothelial marker) and mKitL (green). Nuclei were stained with DAPI (blue). Scale bars, 20 µm. (F) Quantification of mKitL mean fluorescence intensity (MFI) in panel E. (G) In vitro coculture assay with DMSO or Mmp9 inhibitor treatment (5 independent experiments). (H) Hematoxylin and eosin–stained E13.5 FL sections from indicated genotypes, representative of 3 embryos per genotype. Scale bars, 50 µm. Numbers of replicates are indicated at the bottom of each column. Two-tailed Student t tests were used to assess statistical significance. Error bars represent SEM.