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. 2013 Dec 27;8(12):e82777. doi: 10.1371/journal.pone.0082777

Figure 4. Cellular density effect on endocytsis pathways of E-[c(RGDfK)2] and iRGD.

Figure 4

U87 cells were incubated with E-[c(RGDfK)2] at different densities (15000; 50000; 95000 cell/cm2) (A) or iRGD (7000; 35000; 75000 cell/cm2) (B).These densities were defined as low, medium and high density, respectively. Incubations were performed without inhibitor (▪), with nystatin (□), with amiloride (Inline graphic) or with chloroquine (Inline graphic). Results observed after either nystatin or amiloride incubations were not significantly different for all the densities. After chloroquine treatment, significant decreases of E-[c(RGDfK)2] uptake were presented at low, medium and high density (39.8%±2.6%; 38.8±0.8%; 29.5%±2.4%, respectively). Significant iRGD uptake decreases were observed when cells were incubated with chloroquine, 42.4%±1.1% at low density, 48.2±0.2% at medium density and 53.9%±2.5% at high density. The level of autofluorescence of the cells was represented by the dotted line (……). For these experiments (n≥3), the results (☆) and (★,Inline graphic,•,Inline graphic○) were significantly different with p<0.05 and p<0.01, respectively. Insert part B, fluorescent microscopy of cells treated with chloroquine, showed a mainly cytoplasmic membrane signal of iRGD (arrows).