Figure 2.

PMA and CXCL12 promote trafficking of CXCR4 to lysosomes. A and B, HEK293 cells transiently expressing HA-CXCR4-YFP were treated with either vehicle, 100 nm PMA, or 10 nm CXCL12 for 3 h and then coimmunostained with antibodies against LAMP1 for multilabel immunofluorescence microscopy and colocalization analysis. A, representative micrographs of cells treated with vehicle (top), PMA (middle), or CXCL12 (bottom) showing CXCR4-YFP+ puncta (left) and LAMP1+ lysosomes (center) in single-channel and merged images (right). Insets, boxed areas enlarged 5 times. Scale bar, 10 μm. B, quantification of the mean percentage of LAMP1+ lysosomes colocalized with CXCR4-YFP+ puncta in cells treated with vehicle, PMA, or CXCL12. The total number of LAMP1+ lysosomes and colocalization with CXCR4-YFP+ puncta were determined using 3D object-based analysis, as described under “Experimental procedures.” Bars, average from three independent experiments. Error bars, 95.00% CI. The following numbers of cells were used in the analysis: vehicle, n = 32 cells; PMA, n = 21 cells; and CXCL12, n = 31 cells. Data were analyzed using Welch's ANOVA followed by Tamhane's T2 multiple-comparison test. Adjusted p values are indicated.