AZA treatment induces a distinct gene-expression pattern in stromal cells. (A-C) RNA-seq analysis of EL08-1D2 cells after 4 days of culture with or without 10 µM AZA added once on day 0. (A) The term “AZA-sensitive genes” comprises all genes that were differentially expressed between AZA-treated and untreated samples according to DESeq2 analysis. The pie diagram shows the percentage of upregulated vs downregulated differentially expressed genes in the AZA-treated cohort compared with untreated cells. (B) Hierarchical clustering and gene-expression heat map across the 500 most differentially expressed genes between untreated and AZA-treated samples. Values are plotted relative to the average of untreated samples. (C) Overrepresentation of significantly differentially expressed genes in KEGG pathways and GO categories were tested by FNEA implemented in the neaGUI R package.⁴⁰  Entries are ordered first according to false discovery rate (FDR) and second according to the absolute number of the z score. (D-E) Primary healthy and MDS MSCs were treated with AZA once or left untreated and analyzed after 4 days. (D) TGF-β1 expression was validated by comparative RT-PCR in primary MSCs. Results are shown as fold change compared with untreated control. (E) Protein expression of TGF-β1 and TGF-β receptor 1 as assessed by flow cytometry in healthy or MDS MSCs (−/+) AZA. Sample numbers (corresponding to Table 1) of samples analyzed both by RT-PCR in panel D as well as flow cytometry in panel E are indicated. *P < .05; **P < .01; ***P < .001.