Effect of apigenin on LPS-induced proinflammatory cytokines mRNA expression in human THP-1-derived macrophages.

<p>Cells were pretreated with different concentrations of apigenin (A, 6.25, 12.5,25 µM) for 2 h, then treated with LPS (100 ng/mL) for 24 hours. Total cellular RNA was isolated and reverse transcribed. The relative mRNA levels of IL-1β, IL-6, and TNF-α were detected by real-time RT-PCR as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107072#s2" target="_blank">Methods</a>”. Values are mean ± S.E. of three independent experiments. Statistical significance relative to vehicle control, ##p<0.01; ###p<0.001; Statistical significance relative to LPS group, *p<0.05, **p<0.01, ***p<0.001. <b>A</b>. IL-1β; <b>B</b>. IL-6; <b>C</b>. TNF-α.</p

Effect of apigenin on LPS-induced proinflammatory cytokines mRNA expression in mouse J774A.1 macrophages.

<p>Cells were pretreated with different concentrations of apigenin (A, 6.25, 12.5,25 µM) for 2 h and then treated with LPS (100 ng/mL) for 24 hours. Total cellular RNA was isolated and reverse transcribed. The relative mRNA levels of IL-1β, IL-6, and TNF-α were detected by real-time RT-PCR as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107072#s2" target="_blank">Methods</a>”. Values are mean ± S.E. of three independent experiments. Statistical significance relative to vehicle control, ##p<0.01; ###p<0.001; Statistical significance relative to LPS group, **p<0.01, ***p<0.001. <b>A</b>. IL-1β; <b>B</b>. IL-6; <b>C</b>. TNF-α.</p

Effects of apigenin on LPS-mediated mRNA stabilization in mouse J774A.1 macrophages.

<p>Cells were pretreated with apigenin (A, 25 µM) for 2 h and then treated with LPS (100 ng/mL) for 2 h, followed by treatment with actinomycin D (5 µg/ml). Total cellular RNA was isolated at 0, 0.5,1, 2, 4 and 6 h after actinomycin D treatment. The mRNA levels of IL-1β and IL-6 were determined by real-time RT-PCR as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107072#s2" target="_blank">METHODS</a>”. Values are the means ± S.E. from three independent experiments. <b>A</b>. IL-1β; <b>B</b>. IL-6.</p

Effect of apigenin on LPS-induced IL-6 and TNF-α protein expression in in mouse J774A.1 macrophages.

<p>Cells were pretreated with different concentrations of apigenin (A, 6.25, 12.5,25 µM) for 2 h and then treated with LPS (100 ng/mL) for 24 h. At the end of treatment, each cell culture medium was collected. The protein levels of IL-6 and TNF-α were determined by ELISA as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107072#s2" target="_blank">Methods</a>”. Values are mean ± S.E. of three independent experiments. Statistical significance relative to vehicle control, ###p<0.001; Statistical significance relative to LPS group, **p<0.01, ***p<0.001. <b>A</b>. IL-6; <b>B</b>.TNF-α.</p

Effect of apigenin on caspase-1 activation in human THP-1-derived macrophages.

<p>Cells were pretreated with apigenin (A, 6.25, 12.5, 25 µM) for 2 h and then treated with LPS (100 ng/mL) for 24 h. Total cell lysates were prepared for Western blot analysis for caspase-1 protein level as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107072#s2" target="_blank">Methods</a>”. β-Actin was used as a loading control. The relative protein levels of caspase-1 were normalized to β-Actin and analyzed using Odyssey V3.0 software. Values are mean ± S.E. of three independent experiments. Statistical significance relative to vehicle control, ###p<0.001; Statistical significance relative to LPS group, *p<0.05, **p<0.01, ***p<0.001. <b>A</b>. Representative immunoblots of Pro-caspase-1 and active caspase-1 p20. <b>B</b>. The relative protein levels of total caspase-1. <b>C</b>. The relative protein levels of active caspase-1.</p

Effect of apigenin on LPS-induced IL-1β protein expression and maturation in human THP-1-derived macrophages.

<p>Cells were pretreated with apigenin (A, 6.25, 12.5, 25 µM) for 2 h and then treated with LPS (100 ng/mL) for 24 h. The Pro-IL-1β protein level was detected by Western blot analysis. β-Actin was used as a loading control. The mature IL-1β protein level was detected by ELISA as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107072#s2" target="_blank">Methods</a>”. Values are mean ± S.E. of three independent experiments. Statistical significance relative to vehicle control, ###p<0.001; Statistical significance relative to LPS group, ***p<0.001. <b>A</b>. Representative immunoblots of Pro-IL-1β and β-Actin; <b>B</b>. The relative protein levels of pro-IL-1β were analyzed using Odyssey V3.0 software. <b>C</b>. Mature IL-1β level in the media.</p

Effect of apigenin on LPS-induced caspase-1 mRNA expression in mouse J774A.1 macrophages.

<p>Cells were pretreated with different concentrations of apigenin (A, 6.25, 12.5,25 µM) for 2 h and then treated with LPS (100 ng/mL) for 24 hours. Total cellular RNA was isolated and reverse transcribed. The relative mRNA level of caspase-1 was detected by real-time RT-PCR as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107072#s2" target="_blank">Methods</a>”. Values are mean ± S.E. of three independent experiments. Statistical significance relative to vehicle control, ##p<0.01; ###p<0.001; Statistical significance relative to LPS group, **p<0.01, ***p<0.001.</p

Effect of apigenin on pro-inflammatory cytokine-induced activation of NF-κB.

<p>Human 293 Cells were stably transfected with pGL4.32 [luc2P/NF-κB-RE/Hygro] luciferase reporter. Cells were pretreated with apigenin (6.25, 12.5,25 µM) for 2 h and then treated with human IL-1β (10 ng/mL) or TNF-α (10 ng/mL) for 4 h. The luciferase activity was detected as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107072#s2" target="_blank">METHODS</a>”. Values are mean ± S.E. of three independent experiments. Statistical significance relative to vehicle control, ###p<0.001; Statistical significance relative to LPS group, **p<0.01, ***p<0.001. <b>A</b>. IL-1β; <b>B</b>. TNF-α.</p

Effects of apigenin on LPS-mediated mRNA stabilization in human THP-1-derived macrophages.

<p>Cells were pretreated with apigenin (A, 25 µM) for 2 h and then treated with LPS (100 ng/mL) for 2 h, followed by treatment with actinomycin D (5 µg/ml). Total cellular RNA was isolated at 0, 0.5,1, 2, 4 and 6 h after actinomycin D treatment. The mRNA levels of IL-1β and IL-6 were determined by real-time RT-PCR as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107072#s2" target="_blank">METHODS</a>”. Values are the means ± S.E. from three independent experiments. <b>A</b>. IL-1β; <b>B</b>. IL-6.</p

Effect of apigenin on the mRNA and protein expression of ASC and NLRP3 in human THP-1-derived macrophages.

<p>Cells were pretreated with apigenin (A, 6.25, 12.5, 25 µM) for 2 h and then treated with LPS (100 ng/mL) for 24 h. Total cellular RNA was isolated and reverse transcribed. The relative mRNA levels of PYCARD/ASC and NLRP3 were detected by real-time RT-PCR, as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107072#s2" target="_blank">METHODS</a>”. Values are mean ± S.E. of three independent experiments. Statistical significance relative to LPS group, #p<0.05. <b>A</b>. The mRNA level of PYCARD/ASC; <b>B</b>. The mRNA levels of NLRP3. <b>C</b>. The protein levels of ASC and NLRP3 were detected by Western blot analysis as described under “EXPERIMENTAL PROCEDURES”. The representative images of ASC and NLRP3 are shown.</p