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 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

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 AG–4 on cell cycle progression and DNA degradation.

<p>(A) Flow cytometric analysis of cell cycle phase distribution of U937 cells treated with AG–4 (5.4 μM, 0–48 h). The percentage of sub G₀/G₁ cells was assessed using propidium iodide staining. Histograms depict percentage of cells in various phases of cell cycle. The figure is one representative of three independent experiments. (B) Analysis of TUNEL positivity. Control and AG–4 treated (5.4 μM, 48 h) U937 cells were stained as described in Materials and Methods. Cells were examined under light microscope (100X). Presence of DNA nicking is indicated by arrows. The figure is a representative profile of at least three experiments.</p

Andrographolide Analogue Induces Apoptosis and Autophagy Mediated Cell Death in U937 Cells by Inhibition of PI3K/Akt/mTOR Pathway - Fig 8

<p>(A) TEM microphotographs of autophagosomes. Representative electron micrographs of control and AG–4 treated (5.4 μM, 0–48 h) U937 cells. Black arrows indicate autophagosomes and black arrow heads indicate autophagolysosomes including residual digested material. The figure is a representative profile of three experiments. (B) Contribution of autophagy in AG–4 induced cytotoxicity. Cells were pre-treated with 3-MA (10 mM, 4 h) or transfected with siAtg 5 for 72 h followed by treatment with AG–4 (0–50 μM, 48 h). Cell viability was determined by MTS-PMS assay. Results are expressed as IC₅₀ (mean ± SEM) from three independent experiments (***p<0.001, compared to only AG–4 treated cells). (C, D) Effect of anti-oxidant on AG–4 induced autophagy. Cells were treated with AG–4 (5.4 μM, 48 h) in presence or absence of NAC (2.5 mM) followed by flow cytometry for quantification of AVO or immunoblotting for Atg 5 expression levels. (C) Histograms represent the percentage of cells with AVO and have been derived from at least three experiments (***p<0.001, compared to control cells; @@@ p<0.001, compared to only AG–4 treated cells). (D) The figure is a representative profile of at least three experiments.</p

AG–4 induces redox imbalance in U937 cells.

<p>(A) Effect of AG–4 on ROS generation. U937 cells were treated with AG–4 (5.4 μM, 0–3 h), stained with CM-H₂DCFDA and ROS was measured by flow cytometry. For the inhibition of ROS generation, cells were co-treated with NAC (2.5 mM) and AG–4 (5.4 μM, 3 h) and ROS was similarly quantified. Data represent mean GMFC±SEM of three independent experiments (***p<0.001, as compared with control). (B) Effect of antioxidant on survival of U937 cells. Cells were co-incubated with AG–4 (0–50 μM) and NAC (2.5 mM) for 48 h and MTS-PMS assay was performed. Each point corresponds to the mean ± SEM of at least three experiments in duplicate. (C) Effect of antioxidant on AG–4 induced apoptosis. Cells were treated with AG–4 (5.4 μM, 48 h) in presence or absence of NAC (2.5 mM). They were co-stained with Annexin V-FITC and PI followed by analysis for phosphatidylserine externalization using flow cytometry as described in materials and methods. Histograms represent percentage apoptotic cells and have been derived from at least three experiments (***p<0.001, compared to control cells; @@@ p<0.001, compared to only AG–4 treated cells). (D) Effect of AG–4 on level of non-protein thiols. U937 cells treated with AG–4 (5.4 μM, 0–3 h) were labelled with CMFDA and analysed for fluorescence. Data are expressed as mean GMFC±SEM of three independent experiments (*p<0.05, ***p<0.001, as compared with control).</p

Detection of AVO in U937 cells.

<p>Control and AG–4 treated (5.4 μM, 0–48 h) U937 cells in the presence or absence of 3-MA (10 mM, 4 h) were stained with AO (1 μg/ml, 15 min) followed by flow cytometry for quantification or fluorescence microscopy. (A) Microphotograph of AVO. Detection of green and red fluorescence in AO stained cells was performed using a fluorescence microscope (60 X). At least 20 microscopic fields were observed for each sample. (B) Flow cytometric quantification of AVO. Histograms represent the percentage of cells with AVO and have been derived from at least three experiments (*p<0.05, ***p<0.001, compared to control cells; @@@ p<0.001, compared to only AG–4 treated cells).</p

AG–4 induced apoptosis and autophagy are dependent on each other.

<p>(A, C) Effect of inhibitors on AG–4 induced Annexin V positivity and AVO formation. Cells were treated with Z-VAD-fmk (20 μM), 3-MA (10 mM, 4 h) or both Z-VAD-fmk and 3-MA or transfected with siAtg 5 or siBax followed by treatment with AG–4 (5.4 μM, 48 h). (A) Histograms depict percentage of apoptotic cells and are presented as the mean ± SEM from three independent experiments (***p<0.001, as compared with control; @@@p<0.001, as compared with only AG–4 treated cells). (C) Histograms depict percentage of cells with AVO and are presented as the mean ± SEM from three independent experiments (***p<0.001, as compared with control, @@@p<0.001, as compared with only AG–4 treated cells). (B, D) Effect of inhibitors on apoptotic and autophagic proteins. Cells were treated with Z-VAD-fmk (20 μM), 3-MA (10 mM, 4 h) or both Z-VAD-fmk and 3-MA or transfected with siBax or siAtg 5 followed by treatment with AG–4 (5.4 μM, 48 h). Whole cell lysates were prepared and subjected to immunoblot analysis using specific antibodies against Bax or Atg 5. Analysis was confirmed with three different sets of experiments. (E) Effect of simultaneous inhibition of apoptosis and autophagy on AG–4 induced cytotoxicity. Cells were treated with Z-VAD-fmk (20 μM) and 3-MA (10 mM, 4 h) followed by treatment with AG–4 (0–50 μM, 48 h). Cell viability was determined by MTS-PMS assay. Results are expressed as IC₅₀ (mean ± SEM) from three independent experiments (***p<0.001, compared to only AG–4 treated cells).</p

AG–4 induces anti-proliferative effect.

<p>(A) Time-dependent effect in U937 cells. U937 cells were treated with AG–4 (0–50 μM) for 24, 48 or 72 hours. Cell death was assessed by MTS-PMS assay. Each data point represents the mean±SEM of at least three independent experiments in duplicate. (B) Effect on other cells. Human cancer cell lines (Raji, MCF–7, HCT–15) and healthy human PBMC were incubated with AG–4 (0–50 μM) for 48 h. Cell death was assessed by MTS-PMS assay. Each data point represents the mean±SEM of at least three independent experiments in duplicate.</p