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Fig. 1 | Stem Cell Research & Therapy

Fig. 1

From: CXCR7 participates in CXCL12-mediated migration and homing of leukemic and normal hematopoietic cells

Fig. 1

CXCR7 participates in U937 and normal CD34+ cell migration and prevents downregulation of CXCR4 by CXCL12 stimulation. CXCR4 and CXCR7 extracellular and intracellular expression was analyzed in CD34 (a, b) and U937 (c, d) cells on a FACScalibur flow cytometer after labeling with specific antibodies. Results are representative of one experiment of the three to five performed. Histograms show the percentage of cells expressing CXCR4 and CXCR7. The U937 cell line was chosen due to the CXCR4 and CXCR7 expression and high migration capability demonstrated towards CXCL12 (e). Transwell assay shows cell migration toward RPMI + 0.5% BSA, containing or not CXCL12 (200 ng/mL). The number of migrated cells (16 h for U937 and 6 h for CD34+) is expressed as a percentage of the input. The migration of cells was normalized to 100% ± the standard deviation of triplicates. f There was a significant reduction in shCXCR7 U937 cell migration compared to shControl U937 cells. Blocking of CXCR4 by CXCR4 mAb-clone 12G5 promoted a similar effect. Moreover, silencing of CXCR7 plus CXCR4 mAb treatment inhibited cell chemotactic capacity. g The same effect was observed for normal CD34+ cells, i.e., blockage of CXCR7 by CXCR7 mAb-clone 11G8 or CXCR4 or both receptors together also promoted a reduction in cell migration. h shControl and shCXCR7 U937 cells were stimulated with CXCL12, treated or not with phosphatase, and then labeled with anti-CXCR4 UMB-2. This antibody recognizes non-phosphorylated C-terminus. Thus, dephosphorylated samples (using phosphatase) show total CXCR4, whereas untreated aliquots show inactive CXCR4. This figure shows that, in shControl U937 cells, induction with CXCL12 caused activation of CXCR4, since the inactive form does not appear or is very low, which is characteristic of CXCR4 activation (column A). On the other hand, shCXCR7 U937 cells induced by CXCL12 showed no or low expression of total CXCR4 (column D), suggesting that CXCR7 is important for preventing downregulation of CXCR4 in leukemia cells. Anti-transferrin receptor (TfR) was used as control to ensure equal loading. Data represent three independent experiments.*p < 0.05; **p < 0.01; ***p < 0.001; one-way ANOVA and Tukey’s multiple comparison test. Error bars represent standard deviation

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