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

<p>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 () or with chloroquine (). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082777#s3" target="_blank">Results</a> 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 (★,,•,○) 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).</p

MRI charaterization of the cellular density effect on the E-[c(RGDfK)2]-DOTA-Gd internalization.

<p>U87 cells were incubated, for different densities, with E-[c(RGDfK)2]-DOTA-Gd (400 µM). SNR of the different cell densities were significantly higher than the SNR of control (★, p<0.05). The SNR values for the low, medium and high density were not significantly different.</p

Cellular density effect on the cytoskeleton contribution to E-[c(RGDfK)2] and iRGD internalization.

<p>U87 cells were incubated with E-[c(RGDfK)2] at different densities (10000; 40000; 80000 cell/cm²) (A) or with iRGD (10000; 35000; 70000 cell/cm²) (B). These densities were defined as low, medium and high density, respectively. Incubations were performed without inhibitor (▪), with paclitaxel (□), with cytoD () or with colchicine (). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082777#s3" target="_blank">Results</a> with paclitaxel were not significantly different. The cytoD effect on the E-[c(RGDfK)2] internalization was not significant at low density (15.65%±1.3%) but appeared significant for the highest density (23.9%±7.9%). For iRGD, significant results were observed for all the densities when cells were treated with cytoD (29.7%±1.3%; 23.5%±1.3%; 29.0%±2.1% at low, medium and high density, respectively). Treatment with colchicine induced a strong decrease of E-[c(RGDfK)2] and iRGD uptakes. For E-[c(RGDfK)2] the uptake results were 46.3%±1.3%, 46.6±3.0, and 47.5%±3.0% at low, medium and high density, respectively. iRGD uptake results were of 55.0%±0.3%, 53.1%±0.1% and 54.8%±0.8% at low, medium and high density, respectively. For these experiments (n≥3), the results () and (★,,☆,,▾,▵,•,,○) were significantly different with p<0.05 and p<0.01, respectively.</p

Cell density dependence of the U87 cell labeling by E-[c(RGDfK)2] or iRGD.

<p>The «fold increase» represents the normalized value of the median fluorescence intensity (MFI) observed for a given cell density compared to the lowest one (0.8*10⁴ and 10⁴ cell/cm² for the E-[c(RGDfK)2] and the iRGD conditions, respectively). Experimental results were fitted following exponential curves of the type y = a + b × exp^−cx with a, b and c equal to 1.4853±0.064; −0.55979±0.0587; 2.5987×10⁻⁰⁵±7.89×10⁻⁰⁶ and 1.3862±0.0257; −0.52569±0.0249; 2.5936×10⁻⁰⁵±4.42×10⁻⁰⁶ for the E-[c(RGDfK)2] and iRGD respectively.</p

Observation by fluorescent microscopy of the iRGD-alexa488 internalization into U87 cells.

<p>U87 cells were incubated during 1h at 37°C with iRGD-alexa488 at 15 µM (green). Nuclei were stained with DAPI (Blue). Arrows showed vesicular accumulation of the fluorescence inside the cytoplasm. The cytoplasmic membrane labeling was not detectable.</p

Metabolism contribution to E-[c(RGDfK)2] or iRGD internalizations: Effect of the cellular density.

<p>U87 cells were incubated for 1-[c(RGDfK)2] (A) and iRGD (B). Incubations were performed for different media conditions: with Glucose and pyruvate, without oligomycin (condition 1); with Pyruvate, without Glucose and oligomycin (condition 2); without Glucose, pyruvate and oligomycin (condition 3); with Glucose, pyruvate and oligomycin (condition 4); with pyruvate and oligomycin, without Glucose (condition 5); with oligomycin, without Glucose and pyruvate (condition 6). All the experiments were performed at least 3 times. For the E-[c(RGDfK)2] the results for all the conditions (★,,☆,,,,▾,▵) were significantly different with p<0.01. For the iRGD the results for all the conditions (▪,,□,•,,○,♦,,⋄) were significantly different with p<0.01.</p

MRI signal of U87 cells after uptake of E-[c(RGDfK)2]-DOTA-Gd.

<p>U87 cells were incubated at 7*10⁴ cell/cm², with E-[c(RGDfK)2]-DOTA-Gd or MnCL₂ (400 µM during 4 h and 1 h, respectively). Applying a T₁-weighted sequence (T₁-w MRI), results with E-[c(RGDfK)2]-DOTA-Gd were significantly different from the unlabeled cells (★, p<0.05) and from the MnCl₂ condition (•, p<0.05).</p

Experimental determination of oxidation rate, phosphorylation rate and ADP/ATP concentrations.

<p>Our experimental set-up was composed of an oxygraph, a spectrophotometer and a luminometer. An optic fiber, connected to the spectrophotometer, was inserted in the oxygraphic vessel (picture on the top-left hand corner). Mitochondrial oxidation rate was determined using the Clark electrode of the oxygraph. Phosphorylation rate was assessed, with the help of the optic fiber, by the continuous monitoring of NADPH production in the oxygraphic vessel. Samplings were performed at the onset and the end of the recording to assess both ADP and ATP concentrations using a bioluminescence-based assay with the help of a luminometer (picture on the top-right hand corner). For clarity, all parameters that were measured during each experiment are highlighted by colored circles. HK: hexokinase, G6PDH: glucose 6 phosphate dehydrogenase, G6P: glucose 6 phosphate, OM: outer membrane, IM: inner membrane.</p

Typical recording of oxidation rate, phosphorylation rate and ADP/ATP concentrations.

<p>Oxidation and phosphorylation rates were recorded simultaneously in liver and muscle mitochondria oxidizing glutamate+malate+succinate as substrates. Mitochondrial protein concentration in the oxygraphic vessel was 25 µg.ml⁻¹. Steady states of oxygen consumption and phosphorylation rates were obtained using the coupled enzymatic system composed of Glucose (5 mM) - Hexokinase (2.5 U.ml⁻¹, Sigma-Aldrich, H4502) - Glucose-6-phosphotate dehydrogenase (2.5 U.ml⁻¹, Sigma-Aldrich, G6378) - NADP⁺ (1.6 mM). Dashed arrows correspond to the sampling of measurement medium taken from the oxygraphic vessel during each recording for determination of ADP and ATP concentrations.</p

Dependence of oxidation and phosphorylation rates on ADP concentration in liver and muscle mitochondria.

<p>Oxidation and phosphorylation rates were recorded simultaneously in liver (n = 4) and muscle (n = 5) mitochondria oxidizing glutamate+malate+succinate as substrates. True steady state of oxidation and phosphorylation rates were obtained using coupled enzymatic system composed of Glucose (5 mM) - Hexokinase (2.5 U.ml⁻¹, Sigma-Aldrich, H4502) - Glucose-6-phosphotate dehydrogenase (2.5 U.ml⁻¹, Sigma-Aldrich, G6378) - NADP⁺ (1.6 mM). Data presented in panels A and B correspond to absolute oxidation and phosphorylation rates, respectively. Maximal oxidation and phosphorylation rates obtained for muscle and liver mitochondria were 408.0±42.5 vs. 85.2±5.5 nmolO₂.min⁻¹.mg⁻¹ and 1672.8±134.1 vs. 361.3±33.41 nmolATP.min⁻¹.mg⁻¹, respectively. Panels C and D show normalized oxidation and phosphorylation rates, respectively. Data were fitted using the Michaelis-Menten equation presented in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020709#s2" target="_blank">materials and methods</a> section. Data are presented as mean ± SD. Differences were tested using an unpaired bilateral student's t-test. ** p<0.01 between liver and muscle, # p<0.01 vs. KmVox.</p