Fig. 5

Endocytosis and recycling rates of CCR5 surface populations in U87.CD4.CCR5 cells: temperature versus RANTES trigger. U87.CD4 cells were transfected with CCR5-GFP. 24 h post-transfection, cells were divided into groups and treated with or without 100 nM RANTES at 37 °C (A). After 0, 15, 30, 45, 60, 75, or 90 min, cells were fixed and then stained with CTC8 (blue lines), 2D7 (green lines), or 45523 (red lines). Colocalization percentages first were normalized to the 0-min timepoint and graphed. To determine recovery rates, U87.CD4 cells were transfected with CCR5-GFP. 24 h post-transfection, cells were divided into groups and treated with RANTES for 90 min at 37 °C. RANTES was removed, and cells were treated with 1 µM cytochalasin D, 0.45 M sucrose, or left as a volume equivalent control for 0, 30, 60, or 120 min at 37 °C (B). After treatment, cells were fixed and stained with CTC8 (blue lines), 2D7 (green lines), or 45523 (red lines). Colocalization coefficients were calculated using ZEN Blue 2.3 software at ×1.3 zoom using 10 different cells and graphed as a percentage of the control (without treatment)