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 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

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 LPS and malvidin on mitochondrial membrane potential of RAW 264.7 macrophages.

<p>Cells were pretreated or not with malvidin for 30 min and exposed or not to LPS for 1h. Medium was replaced to fresh one without any agents and containing 1 µg/ml JC-1 membrane potential-sensitive fluorescent dye for 15 min. Green and red fluorescence images of the same field were acquired using a fluorescent microscope. Representative merged images of three independent experiments are presented. Con: control; Mv: malvidin.</p

Effect of malvidin, kinase inhibitors and NAC on LPS induced nuclear translocation and DNA binding of of NFκB.

<p>RAW 264.7 macrophages were treated for 1h as indicated, then nuclei were isolated and NFκB was extracted using magnetic beads baited with oligonucleotides of NFκB binding consesus sequence. Total (phosphorylated and unphosphorylated) NFκB (t-p65) was detected by immunoblotting in the samples eluted from the beads. Histon H1 from the isolated nuclei was used as loading control. Representative blots (A) and densitometric evaluations (B) of 3 independent experiments are shown. Pixel densities were normalized to histon H1. Values are given as means ± SEM. * p<0.05, ** p<0.01, *** p<0.001 compared to untreated control, ^# p<0.05, ^## p<0.01, ^### p<0.001 compared to LPS alone. a.u.: arbitrary units; SB203580: p38 MAPK inhibitor; PD98059: ERK inhibitor; NAC: N-acetyl cysteine.</p

Effect of malvidin on LPS induced nuclear translocation and DNA binding of of NFκB.

<p>RAW 264.7 macrophages were treated for 1h as indicated, then nuclei were isolated and NFκB was extracted by using magnetic beads baited with oligonucleotides of the NFκB binding consesus sequence. Total (phosphorylated and unphosphorylated) NFκB (t-p65) as well as the phoshorylated form of its p65 subunit (p-p65) was detected by immunoblotting in the samples eluted from the beads. Histon H1 from the isolated nuclei was used as loading control. Representative blots (A) and densitometric evaluations (B,C) of three independent experiments are shown. Pixel densities were normalised to that of the histon H1. Values are given as means ± SEM. * p<0.05, ** p<0.01, *** p<0.001 compared to untreated control, ^### p<0.001 compared to LPS alone. a.u.: arbitrary units.</p

BGP-15 attenuates hydrogen peroxide-induced mitochondrial reactive oxygen species production in WRL-68 cells.

<p>(A) Effect of hydrogen peroxide and BGP-15 pretreatment (for 30 minutes) on mitochondrial ROS production, as determined by the oxidation of the mitochondrial enriched dye from DHR123 to R123 in WRL-68 cells that had been labelled with mitochondrial directed red fluorescent protein. High magnification fluorescent microscopic images show the different localization of the produced R123. Inserts are expanded from the area indicated by dashed rectangles. (B) Quantification of R123 production. Data are presented as the mean ± SEM of three independent experiments. **P < 0.01 and ***P < 0.001 compared to control cells; ^##P < 0.01 compared to H₂O₂-treated cells.</p