Figure 2. Effect of inhibitors of different cellular uptake pathways on the internalization of fluorescent-labeled MDP by mouse macrophages.

(A) Bone-marrow derived macrophages were treated with the indicated doses of CPZ for 3. 5 hrs and the induction of cell death was evaluated by LDH release. Values represent mean ± SD of triplicate cultures. (B) Percentage of MDP-labeled macrophages after incubation for 15 min with medium alone (-), medium containing MDP-Alexa488 alone (untreated, unt.) or in the presence of CPZ (5 μM), dimethylamiloride (DMA), polyinosinic acid (Poly I) or mannans from Sacharomyces cerevesiae (Mannans). Results are representative of two experiments. (C) Bone-marrow derived macrophages were pretreated or not with CPZ (5 μM) for 30 minutes and then incubated with MDP-Alexa488 or LDL-BODIPY for 60 minutes. The uptake was analyzed by FACS and plotted as percent of uptake inhibition of the untreated cells. Results are presented as the mean of triplicate wells ± SD. Results are representative of two experiments (D) Bone-marrow derived macrophages were pretreated or not with CPZ (5 μM) and stimulated with MDP (10 μg/ml) for the indicated periods of time. Cell lysates were prepared and blotted with indicated antibodies. Results are from one representative experiment of three independent experiments. p, phosphorylated (E) Fluorescence confocal images of mouse macrophages incubated for 3 h with MDP-Rhodamine (red), alone or in the presence of CPZ or Poly I. In all cases, the nuclei were counterstained with DAPI (blue), and cells were fixed and imaged by fluorescence confocal microscopy. Arrows indicate intracellular MDP-Rhodamine *, denotes p < 0.05 between untreated and CPZ-treated cultures. Results are representative of three experiments.