PP242-altered expression of CXCR4 and intracellular signaling in primary AML cells and MSCs cultured alone and cocultured. (Ai) Comparison of CXCR4 expression in primary AML and AML CD34⁺ progenitor cells cultured alone and cocultured with stroma. Results represent the ratio of MFI calculated as described in “Flow cytometry.” P value was calculated using paired 2-sample t test. (Aii) Contour dot plot and histograms demonstrating effects of PP242 on CXCR4 expression in a representative sample. (B) Circos diagram displays the inhibitory effect of PP242 on CXCR4 expression and intracellular signaling in attached AML CD34⁺ cells and corresponding MSC samples in cocultures. The fold inhibition was calculated by MFI of untreated cells divided by MFI of treated cells. I-IV: surface marker and intracellular markers measured by flow cytometry. I: 4EBP1 (cyan), II: pAKT (blue); III: pS6K (purple); IV: CXCR4 (magenta). (A-H) Eight AML CD34⁺ samples and 8 MSCs in cocultures. Left panel shows the attached AML CD34⁺ cells; right panel, their associated MSCs in cocultures. Each ribbon connects a marker and an individual patient. The width of a ribbon represents the degree of inhibition: the wider the ribbon, the greater the degree of inhibition. The color of the ribbon represents an individual marker. (C) The inhibitory effects of short-term PP242 treatment on CXCR4 expression and on mTOR signaling in OCI-AML3 cells. Surface expression of CXCR4, CD44, VLA4 was examined by flow cytometry in OCI-AML3 cells treated with 2.5μM PP242 for the indicated time period. Data represent the MFI ratios (Ci). Histograms of the triplicate experiments measuring changes in CXCR4 levels with 12G5 antibody recognizing the extracellular loop 2 (EC2) domain of CXCR4, and 1D9 antibody recognizing the N-terminal of CXCR4, are shown in panel Cii.