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Molecular Biology of the Cell: E03-09-0634

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Supplemental Figures (PDF)

• Supplemental Figure 1 hCCL3-L1 exposure reduces D6 affinity for ¹²⁵I-hCCL3-L1. HA-D6-expressing HEK293 cells were incubated with or without 200nM hCCL3-L1 for 45 minutes at 37°C, washed, and resuspended in aliquots of 5x10⁵ cells in 50μl of binding buffer (HEK293 medium plus 10mM HEPES (pH7.4)) containing 6nM ¹²⁵I-hCCL3-L1 and a range of unlabelled competitor hCCL3-L1 concentrations. Control samples contained no unlabelled hCCL3-L1. After 90 minutes at 4°C, the cells were collected by centrifugation, and washed with ice cold PBS. Cell-associated radioactivity was then determined. Average uncompeted binding to untransfected cells was subtracted from all counts from receptor-expressing cells, which are then represented as a percentage of the counts obtained in the absence of unlabelled hCCL3-L1. Each point was done in triplicate. The data show that hCCL3-L1 pre-treated HA-D6 cells had a lower subsequent affinity for ¹²⁵I-hCCL3-L1, indicated by a shift in the position of the curve to the right.
• Supplemental Figure 2 Surface D6 levels are not down-regulated after α-D6 internalisation. HEK293 cells expressing HA-D6 were loaded at 4°C for 45 minutes with α-D6 antibody, washed, then either (A) left at 4°C and incubated with secondary antibody, (B) shifted to 37°C for 30 minutes, put back on ice, and incubated with secondary antibody, or (C) shifted to 37°C for 30 minutes, washed, put back on ice and incubated with a further aliquot of α-D6 antibodies for 45 minutes, washed, and then incubated with secondary antibody for 30 minutes. Cells were then assessed by flow cytometry and mean fluorescence intensity determined. Each sample was done in triplicate and the data shown is representative of data generated in repeat experiments. The data demonstrate that despite D6-driven antibody internalisation, surface levels of the receptor are not down-regulated (compare A and C).
• Supplemental Figure 3 In continuous culture, D6-expressing cells more effectively clear ¹²⁵I-hCCL3-L1 than those expressing CCR5. 5x10⁵ cells were plated, grown for 2 days, and fresh medium added containing 50nM hCCL3-L1 and 2nM ¹²⁵I-hCCL3-L1. 60 μl aliquots were taken during this incubation, debris removed (5K rpm, 4°C, 5min), and 50 μl of the supernatant precipitated at 4°C with 12.5% TCA. Degradation was determined as the ratio of TCA precipitable to non-precipitable counts. The low level of degradation by untransfected HEK293 cells (<3% after 24 hrs) was subtracted. Each point shows the mean+/-SD of results from three parallel cultures. On repeat, near identical results were seen. The data show that D6-expressing cells remove ~50% of the radioligand in 12hrs in contrast to the 5% removed by CCR5. After 12hrs, D6 cells continue to degrade ¹²⁵I-hCCL3-L1, whilst CCR5-mediated degradation plateaus. Data from HA-D6 and HA-CCR5 cells is shown, as they have similar levels of resting surface receptor, but CCR5-GFP cells, that have considerably more surface CCR5, also produced a plot like the HA-CCR5 cells (unpublished observation).
• Supplemental Figure 4 β-arrestin-1 (V53D) or dynamin I (K44A) inhibit hCCL3-L1-induced CCR5 internalisation. HEK293 cells expressing CCR5-GFP were transiently transfected with the indicated amounts of plasmids encoding either β-arrestin-1 (V53D) or dynamin I (K44A), as described in Materials and Methods. 24hrs after transfection, cells were harvested and incubated with or without 200nM hCCL3-L1 at 4°C for 30 minutes, shifted to °C for 10 minutes, and returned to 4°C. Cells were then harvested at 4°C by centrifugation, washed with 1.4ml of ice-cold FACS buffer, and CCR5 surface levels assessed by flow cytometry using a PE-coupled α-CCR5 antibody. Mean fluorescence intensity readings were used to determine the percentage reduction in surface CCR5 after hCCL3-L1 treatment of the transfected cells, and these were then compared to the down-regulation seen after hCCL3-L1 treatment of mock-transfected cells (set to 100%). Each bar on the graph shows the mean+/-SD of results from three treatments from each transfection. Longer incubation in hCCL3-L1 showed similar effects of plasmid transfection, and repeated transfections produced similar results. The data show that expression of either β-arrestin-1 (V53D) or dynamin I (K44A) reduce the extent of hCCL3-L1-driven CCR5 internalisation.

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