Gene expression analysis of sorted cells reveals differences between MDS and control HSCs. (A) Unsupervised hierarchal clustering based on gene expression reveals differences between MDS and control HSCs. (B) Volcano plot comparing the difference of mean expression (x-axis) and significance of the difference (y-axis), showing mainly overexpressed genes in MDS HSCs. Red dots indicate P < .05 and fold change > 1 log2. (C) STAT3 gene expression in 183 samples of MDS CD34⁺ cells and 17 healthy controls is shown as a heatmap. (D) Expression is higher in MDS compared with controls (t test with Benjamin-Hochberg correction). Boxplots show the expression of STAT3 in various FAB subtypes of MDS (RCMD is a subset of the FAB RA category). (E) Phosphoflow analysis showing a reduction of pSTAT3 in CD34⁺ BM-derived cells 36 hours after treatment with 0.9μM STAT3 inhibitor V and 100μM STAT3 inhibitor VI. (F) Colony formation of Lin⁻CD34⁺CD38⁻ HSCs derived from patients with MDS (solid bars) and healthy controls (open bars) treated with 0.3 or 0.9μM inhibitor V, 50 or 100μM inhibitor VI, or DMSO showing a significant reduction of MDS colonies when treated with either inhibitor. Shown are averages of colony numbers expressed as percentage of DMSO-treated colony formation (N_MDS = 3; and N_Healthy = 2). Black asterisks represent P values from t tests comparing inhibition of colony formation of MDS with healthy control-derived cells; and gray asterisks, P values from t tests comparing inhibition of colony formation of STAT3 inhibitor-treated versus DMSO-treated cells. *P < .05. **P < .01. (G) Representative pictures of HSC-derived colonies in the presence of STAT3 inhibitor V, VI, or DMSO control. Bars represent 200 μm.