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. 2011 Apr 5;6(4):e18467. doi: 10.1371/journal.pone.0018467

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Saha et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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A: A representative dot plot of uninfected (a) and Leishmania 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 L. donovani infected macrophages (f, ) for DAF-2T that were treated with Berberine chloride (…) macrophages as described in Methods. B: Uninfected macrophages (1×10⁶/ml, □, a) or L. donovani 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 Methods. Data are expressed as the mean GMFC ± SEM of at least 3 experiments in duplicate. C: Uninfected macrophages (1×10⁶/ml, □, a) or L. donovani 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 Methods. Data are expressed as the mean GMFC ± SEM of at least 3 experiments in duplicate. D: Uninfected macrophages (1×10⁶/ml, □, a) or L. donovani 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 Methods. Each point represents the mean ± SEM of NO₂ ⁻ (µM) of at least 3 experiments in duplicate. E: Uninfected macrophages (1×10⁶/ml, □, a) or L. donovani 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 Methods. Each point represents the mean ± SEM of NO₂ ⁻ (µM) of at least 3 experiments in duplicate. F: Uninfected macrophages (a) and L. donovani 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 Methods.

Inline graphic — A: A representative dot plot of uninfected (a) and Leishmania 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 L. donovani infected macrophages (f, ) for DAF-2T that were treated with Berberine chloride (…) macrophages as described in Methods. B: Uninfected macrophages (1×10⁶/ml, □, a) or L. donovani 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 Methods. Data are expressed as the mean GMFC ± SEM of at least 3 experiments in duplicate. C: Uninfected macrophages (1×10⁶/ml, □, a) or L. donovani 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 Methods. Data are expressed as the mean GMFC ± SEM of at least 3 experiments in duplicate. D: Uninfected macrophages (1×10⁶/ml, □, a) or L. donovani 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 Methods. Each point represents the mean ± SEM of NO₂ ⁻ (µM) of at least 3 experiments in duplicate. E: Uninfected macrophages (1×10⁶/ml, □, a) or L. donovani 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 Methods. Each point represents the mean ± SEM of NO₂ ⁻ (µM) of at least 3 experiments in duplicate. F: Uninfected macrophages (a) and L. donovani 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 Methods.