PMC

a,b&d) Representative z-slice confocal images of the same live PBMC T cells or Jurkat T cells expressing the indicated fluorescent fusion proteins (green) and Rab11 (Rab11-RFP; red) ± SDF-1 treatment (n = 6-13). c) Jurkat cells were treated ± SDF-1 for 20 min. Cells were then either stained for CXCR4 surface levels immediately or washed to remove SDF-1 and incubated for 1 hr to allow CXCR4 to recycle back to the cell surface before staining for CXCR4 surface levels. Bars denote the mean results for each condition ± S.E.M.; n = 3; *, significantly different from SDF-1 treated cells without recycling, p < 0.05.

a) Representative z-slice confocal images of a live Jurkat T cell expressing a fluorescent fusion protein of CXCR4-WT (CXCR4-WT-RFP; red) and the MTOC marker, Centrin-2 (Centrin-GFP; green), before and after SDF-1 treatment. Arrows indicate the MTOC (n = 7). b) Representative z-slice confocal images of a live Jurkat T cell expressing CXCR4-WT-YFP (green) and the Golgi marker, GalT-CFP (blue) ± SDF-1 treatment (n = 12). c) Jurkat T cells were stimulated with SDF-1, then fixed and analyzed by electron microscopy. Arrows, Golgi; PM, plasma membrane; M, mitochondrian; N, nucleus; n = 22. d) Endogenous, cell-surface CXCR4 on live Jurkat T cells was labeled with anti-CXCR4-immunogold, cells were treated with SDF-1 for 30 min, then analyzed by electron microscopy. White arrows, Golgi; black arrows, immunogold-CXCR4; n = 6.

a) Jurkat T cells were pretreated with either cytochalasin D (Cyto. D) or vehicle (DMSO), and the endocytosis of endogenous CXCR4 was stimulated by treating cells with SDF-1 for 20 min. Cells were then stained with fluorescently-conjugated CXCR4 mAb and analyzed by flow cytometry (n = 3). b) Jurkat T cells expressing CXCR4-WT-YFP (green) were pretreated with either Cyto. D or vehicle, then assayed as in for CXCR4-WT-YFP endocytosis and trafficking in response to SDF-1. Representative z-slice confocal images are shown. c&d) Results of multiple experiments as in b), quantitated as in . Bars denote the means ± S.E.M. of 4-7 experiments; *, significantly different from SDF-1 - treated control (DMSO) samples, p < 0.05. e) Jurkat T cells expressing CXCR4-WT-YFP and Rab11-RFP, assayed as in b). f) Jurkat T cells were pretreated with either Cyto. D or vehicle (DMSO) and assayed for their ability to recycle endocytosed CXCR4 back to the cell surface as in . *, significantly different from control (DMSO) samples, n = 3, p < 0.05.

a) Jurkat T cells were transiently-transfected with a plasmid encoding both Gα13 shRNA and mCherry, or a control plasmid encoding mCherry. Whole-cell lysates were immunoblotted to assay Gα13 protein levels as compared to Actin (control). b) Jurkat T cells were transiently-transfected as in a) except cells were additionally co-transfected with a plasmid encoding CXCR4-WT-YFP (green). Cells were assayed as in for CXCR4-WT-YFP endocytosis and trafficking in response to SDF-1. c&d) Results of multiple experiments as in b), quantitated as in . Bars denote the means ± S.E.M. of 8 experiments; *, significantly different from SDF-1 - treated control cells, p < 0.05. e) Jurkat cells were transfected with a plasmid encoding Gα13 shRNA as in a), and mCherry+ cells were assayed for endogenous CXCR4 cell-surface levels and SDF-1 -dependent CXCR4 endocytosis as in (n = 3). f) Jurkat cells were transiently-transfected with a plasmid encoding both Gα13 shRNA and eGFP, or a control plasmid encoding eGFP. eGFP+ cells were assayed for recycling of endocytosed CXCR4 as in (n = 3). *, significantly different from control vector-transfected cells.

a) Jurkat T cells were stimulated for 2 min with SDF-1. Active, GTP-bound Rho was affinity purified and detected by SDS-PAGE and immunoblotting (upper gel). Whole cell lysates were analyzed separately as a control (lower gel)(n = 3). b) Jurkat T cells were transfected with either a plasmid encoding RhoN19 or a control vector, together with a plasmid encoding eGFP. GFP+ cells were assayed for CXCR4 cell-surface levels as in (n = 3). c) Jurkat T cells were transiently-transfected with a plasmid encoding CXCR4-WT-YFP, together with either a control plasmid or a plasmid encoding RhoN19, then assayed as in for CXCR4-WT-YFP endocytosis and trafficking in response to SDF-1. Representative z-slice confocal images are shown. d&e) Results of multiple experiments as in c), quantitated as in . Bars denote the means ± S.E.M. of 8 experiments; *, significantly different from SDF-1 - treated control plasmid-transfected cells, p < 0.05.

a) PBMC T cells were stimulated ± SDF-1, then fixed and stained for endogenous CXCR4 using anti-CXCR4-FITC (green). Hoechst staining (blue) and outline (white) show the nucleus and plasma membrane, respectively. Representative z-slice confocal images of 12 individual cells are shown. b) Jurkat T cells were transiently-transfected with plasmids encoding fluorescent fusion proteins of CXCR4-WT, CXCR4-Δc-ter, or CXCR4-KKK/AAA. Confocal microscopy was used to image the same individual live cells before and after 15 min of SDF-1. The images show either a 3-dimensional (3D) image reconstruction or individual z-slice confocal images of representative cells ± SDF-1. Arrows indicate CXCR4-YFP that trafficked into the clustered endosomal compartment. c-e) Results of 3-dimensional image quantitation of multiple experiments as in b), showing for each CXCR4 construct the fraction of total cellular CXCR4-YFP fluorescence present on the plasma membrane (c), the intracellular region (d), or only within clustered endosomes (e). Bars denote the mean values before and after 15 min of SDF-1 ± S.E.M. for CXCR4-WT-YFP, CXCR4-Δc-ter-YFP and CXCR4-KKK/AAA-YFP (n = 22, 4, 9); *, significantly different from unstimulated samples, p < 0.05.