Fig 4. WNT antagonist FRZB is hypermethylated and underexpressed in MDS stroma and treatment of MDS stroma with 5-Aza increases hematopoietic activity.

DNA methylation analysis by HELP-tagging assay shows hypermethylation of selected loci (marked by arrows) in the FRZB promoter in the HS27 stromal cells that are co-cultured with KG1a cells. Dark green denotes CpG islands, while light green denoted CpG shores (A). qRT-PCR shows decreased expression of FRZB in untreated MDS samples (n=4) when compared to control stroma (n=4) or Aza treated MDS (n=2) (T test, P value<0.05) (B). Immunohistochemistry shows increased expression of FRZB in MDS stroma treated with 5-Aza (0.5uM for 5 days) (C). siRNA mediated knockdown of FRZB was achieved in primary MSCs (D). Co-culture with FRZB knockdown MSCs led to increased nuclear β-catenin in CD45+ cells (Representative image shown in E; TTest, P Value <0.01, N=2, F). Healthy CD34+ cells were grown with MDS stromal cells (MDS19 and MDS20) in methylcellulose media. MDS stromal cells (MDS19 and MDS20) that were pretreated with 5-Aza for 5 days led to greater colony formation from healthy CD34 cells, (T test, P value <0.001) (G). Dysplastic colonies seen after co-culture of healthy CD34 cells with MDS stroma (H, left panel). 5-Aza pre-treatment leads to increased size of colonies (H, right panel). FACS analysis of co-cultured cells shows increase in Glycophorin A positive cells in 5-Aza treated MDS stromal co-cultures (n=2, T test, P value<0.5) (I). Increased percentages of all stages of erythroid cells are seen in 5-Aza pretreated stromal co-cultures (J).