ADAM17 co-precipitates with α5β1 integrin and dissociates from the integrin during GPCR stimulation.

<p>(A) Control (−) and 1 µM 5-HT stimulated (+) mesangial cell lysates were immunoprecipitated (IP) with either ADAM17 antibody or Ig control (Ig C), resolved on 3–8% Tris-acetate gel and probed for the presence of β1 integrin and α5 integrin by Western blotting. (B) ADAM17 blot shows that equal amount of ADAM17 were precipitated from each sample. Arrows point to specific and non-specific (NS) bands. One representative example out of four experiments is shown.</p

Manganase²⁺ stimulation decreases association of ADAM17 to β1 integrin and leads to increased ADAM17 activity.

<p>(A) Control and manganese stimulated (1 mM MnCl₂ in PIPES buffer for 2 min) mesangial cell lysates were immunoprecipitated with ADAM17 antibody. Samples were resolved on a 3–8% Tris-acetate gel and probed for β1 integrin and α5 integrin by Western blotting. Immunoblotting for ADAM17 served as loading control. Arrows point to specific and non-specific (NS) bands. One representative blot out of three is shown. (B) Mesangial cells were transfected with AP-HB-EGF construct and stimulated with 1 mM MnCl₂ in PIPES buffer. HB-EGF shedding was expressed as mean±S.D. of fold increase in the rate of change of relative fluorescence units (RFU); **p<0.01 <i>vs</i> control; n = three experiments, 6 parallels/each condition.</p

Co-localization of ADAM17 and α5β1 integrin in rat mesangial cells.

<p>Control (C) and 1 µM 5-HT -stimulated mesangial cell were fixed, permeabilized, and (A) co-immunostained using ADAM17 antibody (green) and β1 integrin antibody (red) as indicated in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033350#s4" target="_blank">Material and Methods</a>”. Arrows indicate co-localization of ADAM17 and α5β1 integrin immunopositive areas (yellow). For the negative controls we omitted the primary antibodies and used PBS followed by secondary antibodies. (B) Parallel samples were incubated with oligonucleotide-labeled PLA probes after incubation with primary antibodies. PLA signals as fluorescence dots were imaged and quantified. As negative control we used either ADAM17 or α5β1 integrin antibody alone followed by the oligonucleotide-labeled PLA probes. Cartoon explains binding of the fluorescence detection reagent only to antibodies in close proximity; **p<0.01. Representative examples out of three experiments are shown.</p

Cell free assays show ADAM17 binding to α5β1 integrin and changes in ADAM17 activity.

<p>(A) Recombinant ADAM17 binds purified α5β1 integrin and recombinant β1 integrin in a cell free binding assay. Plates pre-coated with antibodies against α5β1 integrin or β1 integrin were incubated with purified α5β1 integrin or with β1 recombinant integrin, individually. Recombinant ADAM17 was then added at the indicated concentrations and ADAM17 binding was measured using a colorimetric assay at 450 nm as described in Methods. Data are expressed as mean±S.D. *p<0.05, **p<0.01 <i>vs</i> control; data from 4 experiments with 3 parallels/each condition are shown. (B) Purified α5β1 integrin and recombinant β1 integrin decrease ADAM17 enzymatic activity. Recombinant ADAM17 (10 ng/ml) was incubated alone or together with either purified α5β1 integrin (25 ng/ml or 250 ng/ml) or with β1 recombinant integrin (12 ng/ml or 120 ng/ml), in OG buffer in the presence of a quenched fluorogenic ADAM17 substrate. Enzyme activity was expressed as the rate of change of relative fluorescence units (ΔRFUs⁻¹); *p<0.05, **p<0.01 <i>vs</i> control; data from four experiments with eight parallels/each condition are shown.</p

Integrin expression regulates ADAM17 sheddase activity.

<p>(A) Time-dependent release of alkaline phosphatase (AP)-tagged HB-EGF by unstimulated (C) and 5-HT stimulated cells. Cells were transfected with AP-HB-EGF expressing plasmid and 2 days after transfection they were stimulated with 5-HT for the indicated time. AP activity of cell supernatants was determined using Attophos substrate. Data are expressed as mean±S.D. of fold change in the rate of change of relative fluorescence units; *p<0.05, **p<0.01 <i>vs</i> control at same time point, n = eight experiments, three parallels/each condition. (B) β1 integrin silencing promotes 5-HT-induced AP-HB-EGF shedding. Cells were transfected with AP-HB-EGF expression plasmid together with β1 integrin siRNA (β1-siRNA) or a non-targeting (nt−) siRNA. Successful silencing of β1 integrin was confirmed by resolving the cell lysates on a 4–12% SDS-PAGE and probing for β1 integrin and β-actin (as loading control). Two days after transfection cells were stimulated with 5-HT for 1 h and AP activity of cell supernatants was determined. (C) β1 integrin overexpression inhibits 5-HT induced AP-HB-EGF release in mesangial cells. Cells were transfected with AP-HB-EGF and with β1 integrin expressing plasmid (β1-plasmid) or control DNA (Co-DNA). Successful overexpression of β1 integrin was confirmed by resolving the cell lysates on a 4–12% SDS-PAGE and probing for β1 integrin and β-actin (as loading control). Two days after transfection cells were stimulated with 5-HT for 1 h and AP activity of cell supernatants was determined. Activity data are expressed as mean±S.D. of fold change in the rate of change of relative fluorescence units (RFU); *p<0.05, **p<0.01 <i>vs</i> unstimulated control nt-siRNA or control DNA-transfected cells; ^# p<0.05 and ^## p<0.01 <i>vs</i> 5-HT stimulated nt-siRNA or DNA-transfected cells; n = five experiments, three parallels/each condition.</p

Localization of labeled lipoprotein moieties of oxLDL and oxLDL-IC in lysosomal compartment.

<p>U937 cells were treated with either DiI-labeled lipid moiety (red) (<b>A</b>), or Alexa 546-labeled protein moiety (red) (<b>B</b>) for 90 min and 5 h, with lysosomal marker (Lyso Tracker Green DND-26, 50 nM) applied for the last 30 min of incubation. Cells were treated with labeled oxLDL and oxLDL-IC at 18 µg/ml and 24 µg/ml, respectively. Live cells were washed with DPBS then suspended in sealed capillaries and visualized using confocal microscopy. Arrows point at co-localization of lipid and apolipoprotein moieties of oxLDL and oxLDL-IC with lysosomal compartment.</p

Lipid moiety of oxLDL-IC but not oxLDL co-localizes with induced HSP70/70B' in endosomal vesicles.

<p>RAW 264.7 cells were transfected with HSP70-GFP (<b>A</b>) or HSP70B'-GFP (<b>B</b>), then treated with DiI-oxLDL (24 µg/ml), DiI-oxLDL-IC (32 µg/ml), or DPBS vehicle in serum-free DMEM for 3 h. Alexa 633-transferrin (10 µg/ml) was then added for an additional 2 h. Cells were then fixed with 4% formaldehyde, washed with DPBS, and visualized using confocal microscopy.</p

Differential effect of oxLDL and oxLDL-IC on mitochondrial membrane potential and ROS/RNS production.

<p>U937 cells were grown in phenol red-free IMDM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 50 µg/ml streptomycin, and IFN-γ (200 ng/ml) for 18 h then treated with oxLDL (90 µg/ml), oxLDL-IC, KLH-IC (120 µg/ml) in IMDM up to 5 h. (<b>A</b>) Detection of H₂O₂: Cells were treated with CM-H2DCFDA (5 µM), and Mito Tracker (100 nM) for 30 and 15 min, respectively, prior to conclusion of incubation time with the treatments (5 h). Cells were fixed, suspended in sealed capillaries and visualized using Zeiss LSM 510 Laser scanning confocal microscope. (<b>B</b>) Detection of NO: IFN-γ-treated cells were incubated with L-arginine (100 µM), and DAF-FM diacetate (10 µM) for 1 h, then washed with IMDM twice. Cells were then treated with oxLDL, oxLDL-IC, and KLH-IC as indicated above. Mito Tracker (100 nM) were added 15 min prior to conclusion of incubation time with the treatments (90 min). Live cells were washed, suspended in sealed capillaries and visualized using Zeiss LSM 510 Laser scanning confocal microscope.</p

Localization of labeled lipoprotein moieties of oxLDL and oxLDL-IC in endosomal vesicles.

<p>U937 cells were treated with endosomal marker (Alexa 488-transferrin, green) and with either DiI-labeled lipid moiety (red) (<b>A</b>), or Alexa 546-labeled protein moiety (red) (<b>B</b>) for 90 min and 5 h. Cells were treated with Alexa 488-transferrin (5 µg/ml) and with either labeled oxLDL (24 µg/ml) or labeled oxLDL-IC (32 µg/ml), fixed with 4% formaldehyde, suspended in sealed capillaries and visualized using Zeiss LSM 510 laser scanning confocal microscope. Arrows point at co-localization of lipid and apolipoprotein moities of oxLDL and oxLDL-IC with Alexa 488-transferrin.</p

Lipid moieties of oxLDL and oxLDL-IC co-localize when administered sequentially but not simultaneously.

<p>U937 cells were incubated with DiI-oxLDL-IC (red) and DiO-oxLDL (green) (<b>A</b>) sequentially and (<b>B</b>) in parallel in U937 cells. They were incubated for a total of 5 h. Sequential experiment involved 2 h incubation of oxLDL-IC (32 µg/ml), prior to addition of oxLDL (24 µg/ml) for 3 h. Lyso Tacker Blue DND-22, (50 nM) was used and applied for the last 30 min of incubation. Cells were fixed with 4% formaldehyde, suspended in sealed capillaries and visualized using Zeiss LSM 510 Laser scanning confocal microscope. Arrows point at co-localization of oxLDL in the lysosomal compartment in the upper panel, and the co-localization of oxLDL and oxLDL-IC in the lower panel.</p