EBP is involved in the kE-induced GM₃ levels decrease.

<p><b>A</b>) Fibroblasts were incubated for 2 h with [1-³H]-sphingosine, PBS washed, and radioactive staining of glycolipids was performed for 48 h. Cells were then stimulated with or without 50 µg kE/ml during 5 (dark grey) or 30 min (white). After extraction, lipids were separated by HPTLC and the plates were put in the presence of a radiographic film during one month at −80°C and revealed. GM₃ was identified comparing its migration rate to that of controls. Densitometric analysis: ***, <i>p</i><0.001. <b>B</b>) The radioactive staining and the stimulation have been performed as in A. Lactose (1 mM), an EBP antagonist, was preincubated for 3 h before stimulation with kE (50 µg/ml, 30 min). After extraction, lipids were separated by HPTLC and the plates were put in presence of a radiographic film during one month at −80°C, and revealed. GM₃ was identified by comparing its migration rate to that of controls. Densitometric analysis: NS, not significant; ***, <i>p</i><0.001.</p

LacCer triggers ERK 1/2 activation.

<p><b>A</b>) Fibroblasts were incubated with different concentrations of LacCer for 30 min. Cell extracts were analyzed by anti-phospho-ERK 1/2 (T202/Y204) Western-blot. <b>B</b>) Densitometric analysis: **, <i>p</i><0.01; ***, <i>p</i><0.001.</p

EBP is involved in the kE-induced LacCer production.

<p><b>A</b>) Radioactive staining and stimulation were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014010#pone-0014010-g005" target="_blank">Figure 5</a> caption. LacCer was identified by comparing its migration rate to that of controls. Densitometric analysis: ***, p<0.001. <b>B</b>) Lactose (1 mM) was preincubated for 3 h before stimulation with 50 µg kE/ml for 30 min. After extraction, lipids were separed by HPTLC and the plates were put in presence of a radiographic film during one month at −80°C, and then revealed. LacCer was identified comparing its migration rate to that of controls. Densitometric analysis: ***, <i>p</i><0.001.</p

Exogenous LacCer penetrates into the plasma membrane.

<p>Fibroblasts were incubated with 12.5 µM BODIPY-LacCer (λ_excitation = 505 nm; λ_emission = 511 nm) for 30 min at 4°C followed by washings with defatted-BSA to remove any fluorescent lipid remaining at the plasma membrane as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014010#pone.0014010-Sharma1" target="_blank">[38]</a>. Cells were fixed then analyzed by fluorescence imaging. <b>Hoechst 33342</b>: nucleus staining by Hoechst 33342 (λ_excitation = 340 nm; λ_emission = 465 nm); <b>BODIPY-LacCer</b>: LacCer staining; <b>Merge</b>: superposition of nuclear and 12.5 µM BODIPY-LacCer stainings. The scale represents 10 µm.</p

EBP and lipid rafts are colocalized at the plasma membrane.

<p>EBP and lipid rafts localization were analyzed using confocal microscopy as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014010#s2" target="_blank">Methods</a> section. EBP appears as a red staining, lipid rafts as a green staining and the colocalization as a yellow staining. The scale represents 10 µm.</p

GM₃ plays a substrate role for the elastin receptor complex.

<p><b>A</b>) Fibroblasts were incubated for 30 min with 10 µg/ml anti-GM₃ antibody then treated with 50 µg/ml kE for 30 min. Cell extracts were analyzed by anti-phospho-ERK 1/2 (T202/Y204) and anti-ERK 1/2 Western-blots. The emergence of a third band in ERK1/2 western-blots is due to an electrophoretic shift because of its phosphorylation. <b>B</b>) Densitometric analysis: **, <i>p</i><0.01.</p

Neu-1 is involved in GM₃/LacCer conversion.

<p><b>A</b>) Cellular extracts from untransfected fibroblasts or cells transfected with SNC siRNA or Neu-1 siRNA were analyzed by anti-Neu-1 Western-blot. <b>B</b>) Densitometric analysis: NS, not significant; ***, <i>p</i><0.001. <b>C</b>) Untransfected fibroblasts (control) and fibroblasts transfected with SNC siRNA or Neu-1 siRNA were stimulated with 50 µg/ml of kE. They were stained with or without an FITC-anti-LacCer antibody (1/25 dilution) then analyzed by flow cytometry. The red line represents the control and the green one corresponds to the stimulation with kE (50 µg/ml). <b>D</b>) Fibroblasts were pre-incubated with D-PDMP (20 µM) for 30 min prior to stimulation with kE (50 µg/ml, 30 min). Cell extracts were analyzed by anti-phospho-ERK 1/2 (T202/Y204) and anti-ERK 1/2 Western-blots.</p

Lipid rafts integrity is required for elastin peptide-induced signal transduction.

<p><b>A</b>) Fibroblasts were incubated for 30 min with 10 mM MCD then treated with 50 µg kE/ml for 30 min. Cellular extracts were analyzed by anti-phospho-ERK 1/2 (T202/Y204) and anti-ERK 1/2 Western-blots. <b>B</b>) Densitometric analysis: ***, <i>p</i><0.001.</p

RAP and LRP-1 silencing by siRNA decreased the level and density of lysosome in MCF-7R cells.

MCF-7S, MCF-7R cells that incubated with or without 500 nM RAP for 12 hours were used in this experiment. The endocytic organelles were isolated by density gradient centrifugation as detailed in Materials and Methods. sucrose gradient was analysed using invertase enzyme assay as described in Materials and Methods. Detection of P-gp and Lamp-1 were evaluated by Western-blot in all collected aliquots (A-D). The intensity of the bands was quantified by densitometry using quantity one program. Student’s t-test was used for the statistical significance of different values. ** p < 0.01, *** p < 0.001 for MCF-7R cells versus MCF-7S cells, ## p < 0.01, ### p < 0.001 for MCF-7R treated cells versus MCF-7R untreated cells (E,F,G).</p

Analysis of protein expression and plasma membrane localization of Neu-1 and PPCA.

<p>Young, intermediate and aged fibroblasts were represented by passage 3 (white), 5 (grey) and 8 (black bars). (A) Analysis of Neu-1 and PPCA protein levels during aging. Cells were stimulated or not for 24 hours with 50 μg/mL of EDP before protein extraction. Neu-1 and PPCA were then analyzed by Western blotting using anti-Neu-1 and anti-PPCA antibodies. Densitometric analysis was performed using the Quantity One software. The results were expressed as the ratio Neu-1/β-actin and PPCA/β-actin and normalized to the control expressed in percent. Dotted lines (100%) represent the values of the unstimulated controls. (B) Analysis of Neu-1 and PPCA localization during aging. Neu-1 and PPCA cell localizations were analyzed by confocal microscopy. Bar, 10 μm.</p