CD9 promotes the formation of long, actin-rich cytoplasmic protrusions. (A-B) Immunofluorescence. Cells were incubated for 30 seconds at 37°C with 100 ng/mL CXCL12, subjected to cytospin centrifugation, fixed, and permeabilized. Actin filaments were decorated with rhodamine-phalloidin (1:100) and CD9 was labeled with mouse anti-CD9 antibody (1:50). Confocal imaging of serial Z stacks was performed with a Leica SP5 confocal microscope equipped with a 63×/1.4 oil objective, zoom x3 (A) and ×1(B). The yellow spots on the merged images correspond to the colocalization of actin and CD9. The images shown are representative of 3 independent experiments. (C) The membrane extensions were measured with ImageJ software. The bars indicate the median values. n is the number of cells analyzed: REH CD9^high, n = 34; REH CD9^low, n = 27; ****P < .0001 in Mann-Whitney tests. (D) The distribution of membrane extensions is shown as the percentage of extensions oriented in a range of 10°. n indicates the number of cytoplasmic extensions analyzed: REH CD9^high, n = 34 (black); REH CD9^low, n = 12 (gray).