Figure 2. Activation of NO signaling pathway increases surface CXCR4 expression and HSC homing.
A. Volcano plot of differential expressed genes (DEGs) in Riociguat and vehicle control treated CB CD34+ cells. The list of DEGs can be found in Supplementary Table 3.
B. Selected GO biological processes significantly enriched in DEGs upregulated by Riociguat treatment compared to vehicle control treated group. The number in the box represents the number of activated DEGs involved in the biological process.
C. Heat map of Z-scores (see Supplementary Methods for details) of DEGs associated with “cell migration” upregulated by Riociguat treatment. Each treatment condition has three replicates (r1, r2, r3).
D. Quantitative RT-PCR analysis of CXCR4 expression in vehicle control, SNP, Riociguat or Sildenafil treated CB CD34+ cells. n=3, One-way ANOVA, **p<0.01.
E. Representative histogram of surface CXCR4 expression of human CB CD34+ cells treated with vehicle control, SNP, Riociguat or Sildenafil.
F. Quantification of Mean Fluorescence Intensity (MFI) of membrane CXCR4 expression on CB CD34+ cells treated with vehicle control, SNP, Riociguat or Sildenafil. n=3 CB samples, One-way ANOVA, *p<0.05, **p<0.01.
G. Quantification of Mean Fluorescence Intensity (MFI) of membrane CXCR4 expression on HSCs (CD34+CD45RA−CD38−CD90+CD49f+) treated with vehicle control, SNP, Riociguat or Sildenafil. n=3 CB samples, One-way ANOVA, *p<0.05, **p<0.01.
H. Percentage of human CB CD34+ cells migrated towards human SDF-1, as quantified by flow cytometry. Data pooled from three independent experiments (n=3 CB samples, One-way ANOVA, **p<0.01, N.S. indicates p>0.05).
I. Percentage of human CB HSCs (CD34+CD45RA−CD38−CD90+CD49f+) migrated towards human SDF-1, as quantified by flow cytometry. Data pooled from three independent experiments (n=3 CB samples, One-way ANOVA, **p<0.01, N.S. indicates p>0.05).
J. Percentage of human CD45+ cell chimerism in the BM of NSG mice 24 hours after transplantation with 500,000 CB CD34+ cells treated with vehicle control, SNP or Riociguat. Data pooled from five independent experiments (n=5 mice per group, One-way ANOVA, *p<0.05, **p<0.01).
K. Percentage of human CD45+ cell chimerism in the BM of secondary NSG recipient mice at 16 weeks. n=5 for vehicle control, SNP and Riociguat groups; One-way ANOVA, **p<0.01.
For all panels, data are shown as mean±s.e.m..