Effects of sildenafil treatment on cytokine expressions.

<p>Nitrocellulose based rat cytokine array assay was performed on lung homogenates of untreated (Sham), sildenagfil (Sham+SLD), MCT (PH) and MCT plus sildenafil treated (PH+SLD) rats (n = 3–4). Bar diagrams represent mean±S.E.M. pixel densities of the immunoblots. *** significantly different from Sham group (p≤0.05), +++ significantly different from PH group (p≤0.05) by ANOVA, post hoc Bonferroni test.</p

Effects of sildenafil treatment on NFκB activation and nuclear translocation.

<p>NFκB activation and nuclear translocation was assessed by immunoblotting (A) and immunohistochemistry (B) utilizing phospho-NF-κB p65 (A,B) and NF-κB (A) specific primary antibodies. Bar diagrams (A) of the mean pixel densities±S.E.M. and representative immunohistochemistry images (B) of 3 to 4 animals are presented. The phospho-NFκB p65 bands were normalized to the NFκB band. *** significantly different from Sham group (p≤0.05), +++ significantly different from PH group (p≤0.05) by ANOVA, post hoc Bonferroni test.</p

Effect of sildenafil on ERK1/2 and p38 MAPK activation.

<p>Effect of Sildenafil and MCT treatment on ERK 1 and 2 expression (A) and activation (B) as well as on p38 MAPK expression (C) and phosphorylation (D) was determined by immunoblotting utilizing protein- and phosphorylation specific primary antibodies. The bar diagrams represent pixel volumes±S.E.M. of pERK1/2 and pp38 MAPK bands. The bands were normalized to the appropriate ERK1/2 and p38 MAPK bands. *** significantly different from Sham group (p≤0.05), +++ significantly different from PH group (p≤0.05) by ANOVA, post hoc Bonferroni test. Phospho-ERK1/2 and p38 MAPK are denoted as pERK1/2 and pp38 MAPK.</p

Effect of PARP inhibitor on the PI-3-kinase – Akt pathway and Akt level in transplanted and control kidney samples.

<p>(<b>A</b>) Effects of PARP inhibitor on the Akt1 protein, and phosphorylation of Akt1 and GSK-3 β in control and transplanted kidneys determined using immunoblotting with protein and phospho-specific primary antibodies. Actin was used as loading control. Representative blots of at least three parallel experiments are presented. <b>B</b>. The bar diagrams represent pixel volumes of phosphorylated Akt (serine 473) in kidney samples. The bands were normalized to the appropriate actin band. *p<0.01 transplanted PARP inhibitor treated samples compared to other samples. Difference between control samples (independently of PARP treatment) and transplanted untreated kidney samples were not significant. The bar diagrams represent pixel volumes of GSK-3β (serine 9) phosphorylation bands. The bands were normalized to the appropriate actin band. *p<0.001 transplanted PARP inhibitor treated samples compared to control PARP inhibitor treated, or untreated samples. **p<0.05 untreated transplanted kidney samples compared to control samples (independently of PARP treatment. *** p<0.01 transplanted PARP inhibitor treated samples compared untreated transplanted samples. The bar diagrams represent pixel volumes of Akt1 protein bands. The bands were normalized to the appropriate actin band. *p<0.001 transplanted kidney samples (independently of PARP inhibitor treatment) compared to control kidney samples (independently of PARP inhibitor treatment). (<b>C</b>) Effects of PARP inhibitor on nuclear NF-kappaB and p-NF-kappaB leveles in control and transplanted kidneys determined by immunoblotting with protein and phospho-specific primary antibodies. Actin was used as loading control. Representative blots of at least three parallel experiments are presented. (<b>D</b>) The bar diagrams represent pixel volumes of nuclear NF-kappaB protein bands. The bands were normalized to the appropriate actin band. *p<0.01 transplanted PARP inhibitor treated, or untreated, samples compared to control PARP inhibitor treated, or untreated control samples. **p<0.05 untreated transplanted kidney samples compared to PARP inhibitor treated transplanted kidney samples. The bar diagrams represent pixel volumes of nuclear p-NF-kappaB bands. The bands were normalized to the appropriate actin band. *p<0.001 transplanted PARP inhibitor treated, or untreated, samples compared to control PARP inhibitor treated, or untreated control samples. **p<0.05 untreated transplanted kidney samples compared to PARP inhibitor treated transplanted kidney samples. The vertical axes represent pixel volume means±SEM of the scanned bands of the immunoblots in arbitrary units.</p

Effect of PARP inhibitor on Bcl-2, Bax and t-Bid protein levels in transplanted and control kidney samples.

<p>(<b>A</b>) Effects of PARP inhibitor on the Bcl-2, Bax and t-Bid protein levels in control and transplanted kidneys determined using immunoblotting with protein-specific primary antibodies. Actin was used as loading control. Representative blots of at least three parallel experiments are presented. (<b>B</b>) The bar diagrams represent pixel volumes of Bcl-2 bands. The bands were normalized to the appropriate actin band. *p<0.001 transplanted PARP inhibitor treated samples compared to other samples. The vertical axis represents pixel volume means±SEM of the scanned bands on the immunoblots in arbitrary units. The bar diagrams represent pixel volumes of Bax bands. The bands were normalized to the appropriate actin band. *p<0.01 transplanted PARP inhibitor treated samples, or transplanted untreated samples compared to control untreated and control PARP inhibitor treated samples. ** p<0.05 transplanted PARP inhibitor treated samples compared to transplanted untreated samples. The bar diagrams represent pixel volumes of t-Bid bands. The bands were normalized to the appropriate actin band. *p<0.01 transplanted PARP inhibitor treated samples, or transplanted untreated samples compared to control untreated and control PARP inhibitor treated samples. ** p<0.05 transplanted PARP inhibitor treated samples compared to transplanted untreated samples.</p

Effect of sildenafil on Akt activation.

<p>Effect of Sildenafil and MCT treatment on Akt expression (A) and activation (B) as well as on GSK-3β expression (C) and phosphorylation (D) was determined by immunoblotting utilizing protein- and phosphorylation specific primary antibodies. The bar diagrams represent pixel volumes±S.E.M. of pAkt and pGSK-3β bands. The bands were normalized to the appropriate Akt and GSK-3β bands. *** significantly different from Sham group (p≤0.05), +++ significantly different from PH group (p≤0.05) by ANOVA, post hoc Bonferroni test. Phospho-Akt and GSK-3β are denoted as pAkt and pGSK-3β.</p

Effect of PARP inhibitor on poly-ADP-ribosylation (PAR) and protein nitration in transplanted kidneys.

<p>(A) Immunoblot analysis of poly-ADP-ribosylated HistonH1. HistonH1 was immunoprecipitated with anti-histonH1 antibody, dissolved in sample buffer, subjected to electrophoresis and blotting. Poly-ADP-ribose was detected by anti-PAR antibody. (B) Quantitative analysis of immunoblot samples (C) Representative images of nitrotyrosine immunohistochemistry. Brown color indicates nitrotyrosine positivity; scale bar: 50 µm. (D) Quantitative analysis of immunoblot samples. *: p<0,0001 untreated transplanted kidneys compared to others. ANOVA, Bonferroni post hoc test. Mean±SD. All details described under Materials and Methods.</p

Effect of PARP inhibitor on the structure of tubulo-interstitial system of transplanted kidneys.

<p>Kidneys were fixed in 10% formalin, embedded into paraffin and 5 µm thin sections were cut with microtome. Sections were stained with hematoxylin–eosin (HE). Two representative images of different magnifications are presented for untreated unoperated (Cont.), 4OHQ treated unoperated (Cont.+4OHQ), untreated transplanted (Transp.), and 4OHQ treated transplanted kidneys.Scale bar: 100 and 20 µm for upper and lower row, respectively.</p

Effect of PARP inhibitor on activation and phosphorylation of ERK1/2, JNK1/2 and p38 MAP kinase pathways in transplanted and control kidney samples.

<p>(A) Effects of PARP inhibitor on the ERK1/2, JNK1/2 and p38 MAP kinase phosphorylation and activation in control and transplanted kidneys determined by immunoblotting with phospho-specific primary antibodies. Actin was used as loading control (B) The bar diagrams represent pixel volumes of ERK1/2 phosphorylation bands. The bands were normalized to the appropriate actin band. (p-ERK1) *p<0.001 control PARP inhibitor treated samples compared to control samples. **p<0.01 control PARP inhibitor treated samples compared to transplanted kidney samples independently from PARP inhibitor treatment. ***p<0.05 control untreated sample compared to transplanted kidney samples independently from PARP inhibitor treatment. (p-ERK2) *p<0.001 control PARP inhibitor treated samples compared to control samples. **p<0.01 control PARP inhibitor treated samples compared to transplanted kidney samples independently from PARP inhibitor treatment. ***p<0.05 control untreated sample compared to transplanted kidney samples independently from PARP inhibitor treatment. The bar diagrams represent pixel volumes of p-p38 MAK kinase phosphorylation bands. The bands were normalized to the appropriate actin band. *p<0.001 control samples compared to transplanted kidney samples (independently of PARP inhibitor treatment). **p<0.05 untreated transplanted kidney samples compared to control PARP inhibitor treated transplanted kidney samples. The bar diagrams represent pixel volumes of phosphorylated JNK1/2 bands. The bands were normalized to the appropriate actin band. (p-JNK1) *p<0.05 transplanted untreated kidney samples compared to all others samples. (p-JNK2) **p<0.001 control kidney samples (independently of PARP inhibitor treatment) comparing to transplanted samples. ***p<0.05 transplanted untreated kidney samples compared to transplanted PARP inhibitor treated kidney samples. The vertical axes represent pixel volume means±SEM of the scanned bands of the immunoblots in arbitrary units.</p

Effect of PARP inhibitor on the Bax and Bcl-2 immunohistology in transplanted and control kidneys.

<p>(A) All images show Bax immunohistochemistry, brown color indicates Bax positivity, scale bar: 20 µm. (B). Quantitative analysis of Bax immunohistochemistry samples *: p<0.0001 untreated transplanted kidneys compared to others. ; **: p<0.0001 PARP inhibitor treated transplanted kidneys compared to others. ANOVA, Bonferroni post hoc test. Mean±SD. (C) All images show Bcl-2 immunohistochemistry, brown color indicates Bcl-2 positivity, scale bar: 20 µm. All details described under Materials and Methods. (D) Quantitative analysis of the Bcl-2 immunohistochemistry. *: p<0,0001 transplanted and PARP-treated kidneys compared to others. ANOVA, Bonferroni post hoc test. Mean±SD. All details described under Materials and Methods.</p