Fig. 6

The HD9-10 segment regulates MMP9 gene and protein expression. (a) The location of the gRNAs used to knockout the HD9-10 segment (HD9-10-KO). The HD9-10 segment is the blue bar. The inverted triangles are the nucleotide locations where the gRNA was designed to cut, and the colors of the triangles correspond to the different fluorescent labels, MFP488 (green) or Cy5 (red). Created with BioRender.com. (b) The HD9-10 knockout cell lines (HD9-10-KO_c1 and HD9-10-KO_c2) have significantly decreased MMP9 gene expression compared to the WT cell line. (c) Images and (d) quantification of Western blots showing MMP9 protein expression was significantly reduced in the HD9-10-KO_c1 and HD9-10-KO_c2 cell lines compared to the WT cell line. Details of antibody concentration and cell counts used for western blots are provided in Supplementary Table S9. (e) Two ARID3A binding sites were identified in HD9-10. The ARID3A binding motif is from the JASPAR database⁴⁴. (f) The ARID3A binding sites were each deleted from the E1-pGL4.23 vector (ARID3A_1-del and ARID3A_2-del), and luciferase assays showed significantly decreased activity compared to E1. The y-axis shows enrichments relative to the pGL4.23 vector (empty vector control). The black dots in (b), (d) and (f) are individual biological replicate values for the experiment. (*) indicates p-value < 0.05, and (***) indicates p-value < 0.001. All experiments were performed with three biological replicates. Results are displayed as the mean +/- standard error (SE) and p-values calculated using a two-tailed student’s t-test.