CD9 increases RAC1 activation through its cytoplasmic carboxy-terminal sequence. (A) REH cells were incubated with 1 μM CD9 or scrambled permeant (SCR) peptide for 2 hours. Cells were then subjected to cytospin centrifugation and fixed with 4% paraformaldehyde. The incorporation of fluorescent peptides was monitored with the DeltaVision Epifluorescence Microscope Imaging System. (B-D) REH cells were incubated for 2 hours with 1 μM CD9 or scrambled permeant peptide and were stimulated for 30 seconds with CXCL12 (100 ng/mL). (B) RAC1 activation was analyzed by immunoprecipitation, followed by western blotting for RAC1. The intensity of the signals on the blot was determined with ImageJ software. The graph shows the means ± SD of 3 independent experiments. **P < .01 *P < .05 in Student t test. (C) Cells were subjected to cytospin centrifugation, fixed in 4% paraformaldehyde, and labeled with mouse anti-CD9 antibody (1:50). Confocal imaging on serial Z stacks was performed with a Leica SP5 confocal microscope equipped with a 63×/1.4 oil objective. The membrane extensions were measured with ImageJ software. The bars indicate the median values for each set of conditions. n is the number of cells analyzed: SCR peptide, n = 19; CD9 peptide, n = 45 cells; ****P < .0001 in a Mann-Whitney test. (D) The distribution of membrane extensions is represented according to the percentage of extensions oriented in a range of 10°. n is the number of cytoplasmic extensions analyzed: SCR peptide, n = 36, CD9 peptide, n = 33. (E) Model for CD9 regulation of RAC1 activation and chemotactic migration in B-ALL.