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 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

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 LPS and malvidin on MKP-1 expression in LPS treated RAW 264.7 macrophages.

<p>Effect of LPS and malvidin on steady state MKP-1 protein level was assessed by immunoblotting from whole cell lysate after treating the cells as indicated for 1h. Actin was used as a loading control. Representative blots (A) and densitometric evaluations (B) of 3 independent experiments are shown. Pixel densities were normalized to that of the actin. MKP-1 mRNA expression (C) was determined in another aliquot of cells treated as above using Q-RT-PCR analysis. β-Actin was used as a housekeeping control gene. Specific primer sequences and PCR conditions are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065355#s2" target="_blank">Materials and Methods</a>. Values are given as means ± SEM. * p<0.05, *** p<0.001 compared to untreated control, ^## p<0.01 ^### p<0.001 compared to LPS alone.</p

Effect of malvidin on LPS induced activating phosphorylation of NFκB.

<p>Total (phosphorylated and unphosphorylated) NFκB (t- NFκB) as well as the phoshorylated form of its p65 subunit (p-NFκB) was detected by immunoblotting of whole RAW 264.7 macrophage lysates after treating the cells for 1h as indicated. Actin was used as a loading control. Representative blots (A) and densitometric evaluations (B,C) of 3 independent experiments are shown. Pixel densities were normalized to that of the actin. Values are given as means ± SEM. *** p<0.001 compared to untreated control, ^### p<0.001 compared to LPS alone.</p

Effect of malvidin on LPS induced activation of ERK, p38, JNK MAPK in RAW 264.7 macrophages.

<p>Steady state phosphorylation of ERK, p38 and JNK was detected by immunoblotting from whole cell lysate after treating the cells as indicated for 1h. Actin was used as a loading control. Representative blots (A) and densitometric evaluations (B–D) of 3 independent experiments are shown. Pixel densities were normalized to that of the actin. Values are given as means ± SEM. ** p<0.01, *** p<0.001 compared to untreated control, ^# p<0.05, ^### p<0.001 compared to LPS alone.</p

Effect of malvidin on LPS induced activation of Akt1 in RAW 264.7 macrophages.

<p>Steady state level of total (phosphorylated and unphosphorylated) Akt1 (t-Akt) as well as phosphorylation of Akt1 and its down-stream target GSK-3β was detected by immunoblotting from whole cell lysate after treating the cells as indicated for 1h. Actin was used as loading control. Representative blots (A) and densitometric evaluations (B–C) of 3 independent experiments are shown. Pixel densities were normalized to actin. Values are given as means ± SEM. *** p<0.001 compared to untreated control, ^### p<0.001 compared to LPS alone.</p

Effect of malvidin on LPS induced activation of NFκB in RAW 264.7 macrophages.

<p>Cells were pretreated with 0–100 µM malvidin (black bars in B) or 0–100 µM trans-resveratrol (gray bars in B) for 30 min as indicated. Activation of NF-κB was assessed by a luciferase (A) or an alkaline phosphatase (B) reporter assay after 1 µg/mL LPS exposion for 24 h. Values are given as means ± SEM of 4 independent experiments running in 3 parallels. ** 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.</p