Chs is internalized mainly by passive diffusion.

<p>Panel (A) shows the incorporation of intracellular radioactivity as a function of time when RAW cells were exposed to Chs-LDL (×); Chs-(Ac-LDL) (□); and Chs-POPC liposomes (○), with similar ³H-Chs levels. After incubation the cells were acid washed and then scraped, and finally the radioactivity and protein were quantified as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034822#s4" target="_blank">Methods</a>. Panel (B) shows the effect of specifically blocking the scavenger receptors CD36, SRBI and CD204 by first exposing the RAW cells to specific inhibitory antibodies for 15 min on ice followed by raising the temperature to 37°C and exposing the cells to ³H-Chs-LDL, ³H-Chs-(Ac-LDL), both at 300 ug/ml Apo-B, or ³H-Chs-POPC liposomes. The Chs concentration and the ³H radioactivity was similar in all Chs-containing particles. Results are expressed as percentages relatively to the control cells (treated similarly but with non-inhibitory antibodies). After incubation with the Chs-containing particles the cells were processed as described above. Chs uptake results were expressed as percentages relative to the control. The results are the mean ± SD of three independent experiments. **, p<0.01; *, p<0.05. Panel (C-<b>C^II</b>) shows that RAW cells pre-incubated with Chs-POPC liposomes during 16 h and subsequently exposed to Nat-LDL accumulate neutral lipids in the lysosomal compartment. RAW cells were incubated with Chs-POPC liposomes during 16 h and then with Nat-LDL (300 µg/ml) for 48 h in absence of liposomes. The cells were then fixed and double labeled with Bodipy (C<b>^I</b>) for neutral lipid staining and anti-LAMP-2 antibody (C<b>^II</b>). The merged image (C) shows Bodipy staining in green and LAMP-2 staining in red. Arrows point to LAMP-2-positive cytosolic structures that contain neutral lipids. Bar, 10 µm. Panels (D–<b>G</b>) show that the lysosomal accumulation of neutral lipids induced by pre-treatment of RAW cells with Chs-LDL is irreversible. Raw cells were pulsed with 300 µg/ml of Ac-LDL (panel <b>D</b>) or Chs-LDL (panel <b>F</b>) for 48 h and then chased for 96 h. Cells pulsed with Ac-LDL (D) and then chased (E). Cells pulsed with Chs-LDL (F) and then chased (G). Lipid-rich structures visualized by Oil-red staining (red), DAPI staining (blue). All are merged images. Bars, 10 µm. The total volume of lipid structures per cell, quantified after the 96 h chase, is shown in panel (H). The results are the mean ± SD of three independent experiments. In every experiment 20 individual cells were analyzed. ***, p<0.0001; *, p<0.05; ns, not significant.