FIGURE 4.

Palmitate induces DR5 receptor clustering within the plasma membrane. Huh-7 cells were transfected with DR5-EGFP (A, B, and D) or DR4-EGFP plasmid (A, C, and D) for 36 h. A, total cellular EGFP fluorescence was visualized by digitized fluorescent microscopy. Both individual cellular EGFP fluorescence and EGFP-positive cells were quantified using imaging software to demonstrate that DR5-EGFP or DR4-EGFP plasmids transfected into the cell with the same efficiency. Data are expressed as means ± S.E. for three experiments. B and C, cells transfected with DR5-EGFP or DR4-EGFP for 36 h were treated with PA at 800 μm for 0, 30, 60, and 120 min. D, cells transfected with DR5-EGFP or DR4-EGFP for 36 h were treated with FLAG-tagged TRAIL (400 ng/ml) for 0 and 30 min. FLAG-tagged TRAIL was pre-oligomerized using the M2 monoclonal anti-FLAG antibody (2 μg/ml) as described previously (21). Cells were then fixed and EGFP fluorescence localized at the plasma membrane was analyzed by TIRF microscopy. Representative images are depicted for each condition. The percentage of cells with EGFP clusters was quantified and is expressed as means ± S.E. for three experiments. EGFP fluorescent intensity per cell was also quantified using image analysis software and is expressed as mean ± S.E. for three experiments; *, p < 0.05, RFU, relative fluorescent units.