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

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

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

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

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

Combined ethanol and cholesterol feeding stimulates stellate cell activation and alters TGF-β signaling in the liver.

<p>Mice were fed CTR, Chol, EtOH and EtOH+Chol diets for 3 months. <b>A</b>, representative immunoblots of α-SMA, TGF-β1, BAMBI, Smad2/3, phospho-Smad2/3 and actin. <b>B</b>, quantification of α-SMA immunoblots by densitometry. <b>C</b>, quantification of TGF-β1 immunoblots. <b>D</b>, quantification of BAMBI immunoblots. <b>E</b>, detection of miR33a by RT-qPCR. <b>F</b>, quantification of phospho-Smad2/3 immunoblots. Values are means ± S.E.M. <b>a</b>, p < 0.05 vs CTR; <b>b</b>, p < 0.05 vs Chol; <b>c</b>, p < 0.05 vs EtOH (n = 4 per group).</p

Combined ethanol and cholesterol feeding increases hepatic triglycerides and cholesterol but suppressed fatty acid synthase and carnitine palmitoyltransferase-1 expression.

<p>Mice were fed CTR, Chol, EtOH and EtOH+Chol diets for 3 months. Triglycerides (<b>A</b>) and cholesterol (<b>B</b>) in liver extracts were measured using commercial kits. FAS and Cpt1 were detected by immunoblotting (<b>C</b>, representative images). <b>D</b>, quantification of FAS immunoblots by densitometry. <b>E</b>, quantification of Cpt1 immunoblots. Values are means ± S.E.M. <b>a</b>, p < 0.05 vs CTR; <b>b</b>, p < 0.05 vs Chol; <b>c</b>, p < 0.05 vs EtOH (n = 3–4 per group).</p

Combined ethanol and cholesterol feeding enhances inflammatory responses.

<p>Mice were fed CTR, Chol, EtOH and EtOH+Chol diets for 3 months. <b>A</b>, representative immunoblots of HMGB-1, TLR4, TNFα, ICAM-1 and actin. <b>B</b>, quantification of HMGB-1 immunoblots by densitometry. <b>C</b>, quantification of TLR4 immunoblots. <b>D</b>, quantification of TNFα immunoblots. <b>E</b>, quantification of ICAM-1 immunoblots. Values are means ± S.E.M. <b>a</b>, p < 0.05 vs CTR; <b>b</b>, p < 0.05 vs Chol; <b>c</b>, p < 0.05 vs EtOH (n = 4 per group).</p

Combined ethanol and cholesterol feeding exacerbates liver injury and steatosis.

<p>Mice were fed CTR, Chol, EtOH or EtOH+Chol diets for 3 months. <b>A</b>, representative images from H&E stained liver sections. <b>B</b>, serum alanine aminotransferase (ALT). <b>C</b>, representative immunoblots of cleaved caspase-3 (CC3) and actin. <b>D</b>, quantification of CC3 immunoblots by densitometry. Values are means ± S.E.M. <b>a</b>, p < 0.05 vs CTR; <b>b</b>, p < 0.05 vs Chol; <b>c</b>, p < 0.05 vs EtOH (n = 4 per group).</p

Combined ethanol and cholesterol feeding decreases metalloproteinase expression in the liver.

<p>Mice were fed CTR, Chol, EtOH and EtOH+Chol diets for 3 months. <b>A</b>, representative immunoblots of MMP-2, MMP-9 and actin. <b>B</b>, quantification of MMP-2 immunoblots by densitometry. <b>C</b>, quantification of MMP-9 immunoblots. Values are means ± S.E.M. <b>a</b>, p < 0.05 vs CTR; <b>b</b>, p < 0.05 vs Chol; <b>c</b>, p < 0.05 vs EtOH (n = 4 per group).</p