Anti amastigote activity and safety index of Berberine chloride.

<p>The anti-leishmanial activity of Berberine chloride (0–25 µM, 72 h) was tested in intracellular amastigotes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. Each point corresponds to the mean ± SD of at least three experiments in duplicate. <b>Inset</b>: The effect of Berberine chloride (0–100 µM) on viability of murine macrophages was evaluated at 48 h (▪), 72 h (▴) and 96 h (▾) by the MTS-PMS assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. Each point corresponds to the mean ± SD of at least three experiments in duplicate.</p

Effect of Berberine chloride on IL-12p40 in macrophages.

<p><b>A:</b> Uninfected (a) and <i>L. donovani</i> infected (d) macrophages were treated for 18 h with Berberine chloride 2.5 µM (b, e) or 10 µM (c, f). RNA was isolated, subjected to RT-PCR and the products of β-actin and IL-12 p40 mRNA were resolved on an agarose gel (1.5%) and quantified densitometrically using Total lab software as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. <b>B:</b> Uninfected macrophages (1×10⁶/ml, □, a) or <i>L. donovani</i> infected macrophages (▪, a) were treated with Berberine chloride 2.5 µM (b) and 10 µM (c) for 24 h and assayed for levels of IL-12p40 in culture supernatants by ELISA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. Each point represents the mean ± SEM of IL-12p40 (pg/ml) of at least 3 experiments in duplicate.</p

Effect of Berberine chloride on MAPK pathway in macrophages.

<p><b>A:</b> A representative profile of uninfected macrophages was treated with Berberine chloride (10 µM) for 30 min-6 h. The cells were lysed and subjected to Western blotting with anti-pERK1/2 (a), anti-pp38 MAPK (b) and anti-ERK1/2 (c) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. <b>B:</b> A representative profile of <i>Leishmania</i> infected macrophages were treated with Berberine chloride (10 µM) for 30 min-6 h. The cells were lysed and subjected to western blotting with anti-pERK1/2 (a), anti-pp38 MAPK (b) and anti-ERK1/2 (c) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>.</p

Effect of Berberine chloride on generation of NO and expression of iNOS.

<p><b>A:</b> A representative dot plot of uninfected (a) and <i>Leishmania</i> infected (d) murine peritoneal macrophages, that were treated with Berberine chloride (10 µM, 48 h, b, e). Cells were gated on the basis of characteristic linear forward and side scatter features of macrophages and subsequently DAF-2T fluorescence was measured on a logarithmic scale in the FL1 channel. A representative histogram of uninfected macrophages (c, ) and <i>L. donovani</i> infected macrophages (f, ) for DAF-2T that were treated with Berberine chloride (…) macrophages as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. <b>B:</b> Uninfected macrophages (1×10⁶/ml, □, a) or <i>L. donovani</i> infected macrophages (▪, a) were treated for 24 h with Berberine chloride 2.5 µM (b) and 10 µM (c), and processed for measurement of DAF-2T fluorescence as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. Data are expressed as the mean GMFC ± SEM of at least 3 experiments in duplicate. <b>C:</b> Uninfected macrophages (1×10⁶/ml, □, a) or <i>L. donovani</i> infected macrophages (▪, a) were treated for 48 h with Berberine chloride 2.5 µM (b) and 10 µM (c) and processed for measurement of DAF-2T fluorescence as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. Data are expressed as the mean GMFC ± SEM of at least 3 experiments in duplicate. <b>D:</b> Uninfected macrophages (1×10⁶/ml, □, a) or <i>L. donovani</i> infected macrophages (▪, a) were treated for 24 h with Berberine chloride 2.5 µM (b) and 10 µM (c) and assayed for levels of extracellular NO as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. Each point represents the mean ± SEM of NO₂⁻ (µM) of at least 3 experiments in duplicate. <b>E:</b> Uninfected macrophages (1×10⁶/ml, □, a) or <i>L. donovani</i> infected macrophages (▪, a) were treated for 48 h with Berberine chloride 2.5 µM (b) and 10 µM (c) and assayed for levels of extracellular NO as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. Each point represents the mean ± SEM of NO₂⁻ (µM) of at least 3 experiments in duplicate. <b>F:</b> Uninfected macrophages (a) and <i>L. donovani</i> infected macrophages (d) were treated for 18 h with Berberine chloride 2.5 µM (b, e) or 10 µM (c, f). RNA was isolated and subjected to RT-PCR and the products of β-actin and iNOS mRNA were resolved on an agarose gel (1.5%) and quantified densitometrically using Total lab software as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>.</p