Figure 7
A high level of CCL3 suppresses erythroid differentiation of HSPCs via activation of the CCL3/CCR1/p38 signalling pathway in MM. (A) Flow cytometry analysis showing the expression of CCL3 receptors (CCR1, CCR4 and CCR5) in CD34+ cells from normal donors and NDMM patients. (B) The CFC assay showed the BFU-E colony-forming ability of CD34+ cells in the presence or absence of CCL3 (50 ng/ml) and the CCR1 antagonist BX471 (1 μM), the CCR4 antagonist AZD2098 (100 nM), or the CCR5 antagonist maraviroc (15 nM) (left graphs). The histograms indicate the absolute number of BFU-E colonies formed (right). (C) Immunofluorescence staining of phosphorylated p38 (orange) and GATA1 (green) in EPO-induced HSPC (CD34+) differentiation in the presence or absence of CCL3/BX471. Nuclei were stained with DAPI. Fluorescence images viewed under a confocal microscope were acquired. The scale bar indicates 10 μm. (D) RT-PCR analysis showed the expression levels of GATA1 and KLF1 in CD34+ cells in the presence or absence of CCL3/BX471. GAPDH expression was used as an internal control. (E) Western blotting analysis of phosphorylated p38 (phos-p38), total p38 and GATA1 after the induction of erythroid differentiation in the presence or absence of CCL3/BX471. GAPDH expression was used as an internal control.
