CD9 downregulation impairs CXCL12-induced migration by modulating RAC1 activation. (A) REH CD9^high, CD9^medium, and CD9^low cells were stimulated with CXCL12 (100 ng/mL) for 30 seconds at room temperature. RAC1-GTP levels were analyzed by immunoprecipitation followed by western blotting for RAC1. Antibody binding was detected by chemiluminescence (Immobilon kit; Western-Millipore), viewed with the ImageQuant Fuji LAS 4000 Mini (GE Health Biosciences) acquisition system. The intensity of the signals obtained on the western blot was determined with ImageJ software. The graph shows the mean values ± standard deviation (SD) for 3 independent experiments. ***P < .001 in Student t test. (B) RAC1 activation (RAC1-GTP/RAC1 total) was analyzed according to CD9 expression level (MFI for CD9) in blasts extracted from the BM of B-ALL patients collected on diagnosis (n = 8 patient samples; r = 0.79 P = .0279 in Spearman rank correlation test). (C) REH cells were incubated with NSC23766 (10 μM and 25 μM) for 3 hours at 37°C and stimulated with CXCL12 (100 ng/mL) for 30 seconds at room temperature. RAC1 activation was analyzed by immunoprecipitation followed by western blotting. The migration rates are plotted using a scatter dot plot. The histograms represent the means of 6 independent experiments. *P < .05 in Wilcoxon test. The migration of the cells in response to CXCL12 (100 ng/mL) was also assessed in a Boyden chamber. The graph represents the migration rates obtained in each condition using a scatter dot plot, with the histograms indicating the means of 6 independent experiments. *P < .05 in Wilcoxon test. (D) REH CD9^high, CD9^medium, and CD9^low cells were treated with 10 μM NSC23766 before the assay. The migration of cells in response to a gradient of CXCL12 (100 ng/mL) was assessed in a Boyden chamber, on the basis of CD9 expression. Results are expressed as migration rates and represented using a scatter dot plot. The histograms indicate the means of 6 independent experiments. *P < .05 in Wilcoxon test. (E) REH cells were transduced with 2 shRNAs targeting RAC1 mRNA. RAC1 and CXCR4 protein levels were determined by western blotting. (F) Survival analysis. Cells were injected IV (10⁵ cells) into 4-week-old NSG mice. The general condition of the mice was monitored daily until their death. Kaplan-Meier survival curves were plotted. n is the number of mice used: REH RAC1^high, n = 7; REH RAC1^low no. 1, n = 6; REH RAC1^low no. 2, n = 6; *P < .05 ***P < .001 in log-rank (Mantel-Cox) test.