Expression of Autophagy proteins in U937 cells.

<p>(A) Cells were treated with F2 (18.6 µg/ml for 6 h, 12 h, 24 h, 48 h) and the expressions of Beclin-1, Atg 3, Atg 5, Atg 7, Atg 5-Atg 12 were analyzed by western blotting in whole cell extracts of control and treated cells. Analysis was confirmed with three different sets of experiments. β-actin served as a loading control. (B) Conversion of LC3-I to LC3-II. Whole cell lysates of control and F2 treated (18.6 µg/ml; 0–48 h) U937 cells were prepared and subjected to immunoblot analysis of expression level of LC3-I & LC3-II using anti LC-3 antibodies. β-actin was used to ensure equal loading. The figure is a representative profile of at least three experiments. (C) Densitometric study. Densitometric quantification of bands obtained by immunoblotting was done and LC3 protein expression was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071672#s2" target="_blank">Materials and methods</a>. Histograms represent LC3 protein expression normalized to β-actin (**p<0.01). (D) Contribution of autophagy in F2 induced cytotoxicity. Cells (2.5×10⁴/100 µl of RPMI 1640 medium/well) were pre-incubated with autophagy inhibitor 3-MA (10 mM) or both 3-MA (10 mM) and Z-VAD-FMK (20 µM) for 4 h before the addition of F2. Cell viability was evaluated by MTT assay after treatment with 0–100 µg/ml F2 for 48 h as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071672#s2" target="_blank">Materials and methods</a>. Histograms represent percentage cell viability (Mean ± SEM) obtained at the IC₅₀ concentration of F2 (18.6 µg/ml) and has been derived from at least three experiments in duplicate (***p<0.001 compared to only F2 treated cells).</p

DNA fragmentation and morphological changes induced by F2.

<p>Control and F2 (18.6 µg/ml; 0–48 h) treated U937 cells (2.5×10⁵/ml) stained with Hoechst 33258 were observed under a Leica confocal microscope (100X). The figure is a representative profile of at least three experiments.</p

Cytotoxicity of fractions of n-butanol extract of flowers of <i>Sesbania grandiflora.</i>

<p>Log phase cells (1.25–2.5×10⁴/100 µl of RPMI 1640 medium/well) were treated with fractions (F1, F2, F3, F4) of n-butanol extract of <i>Sesbania grandiflora</i> (0–50 µg/ml) for 48 h, and cell viability was measured by MTT assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071672#s2" target="_blank">Materials and methods</a>. Each IC₅₀ (Mean ± SEM) has been derived from at least three experiments in duplicate.</p

(A) Effect of anti-oxidant on F2 induced autophagy.

<p>Cells were treated with F2 (18.6 µg/ml; 48 h) with or without pre-incubation using NAC (2.5 mM; 3 h). Whole cell lysates were prepared and subjected to immunoblot analysis using specific antibodies against LC3. The figure is a representative profile of at least three experiments. (B) Effect of 3-MA on F2 induced Annexin V positivity. Cells were treated with F2 (18.6 µg/ml; 48 h) with or without pre-incubation using 3-MA (10 mM; 4 h). They were co-stained with PI and Annexin V-FITC followed by analysis using flow cytometry as described before. Histograms represent percentage of apoptotic cells and have been derived from at least three experiments (***p<0.001 compared to control cells). (C) Effect of 3-MA and Z-VAD-FMK on apoptosis and autophagy. Cells were treated with F2 (18.6 µg/ml; 48 h) with or without pre-incubation using 3-MA (10 mM; 4 h) or Z-VAD-FMK (20 µM; 4 h). Whole cell lysates were prepared and subjected to immunoblot analysis using specific antibodies against Bax or LC3. Analysis was confirmed with three different sets of experiments.</p

Involvement of mitochondrial pathway in F2 induced apoptosis.

<p>(A) Loss of mitochondrial membrane potential. U937 cells (2.5×10⁵/ml) were incubated with F2 (18.6 µg/ml for 6 h, 12 h, 24 h, 48 h). Cells were loaded with mitochondrial sensor dye JC-1 (7.5 µM; 15 min) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071672#s2" target="_blank">Materials and methods</a> and analysis was revealed by the shift from red to green fluorescence in time dependent manner. Data is a representative of three different experiments. (B) The expression levels of pro- and anti-apoptotic proteins. Whole cell extracts were made from control and F2 treated U937 cells (2.5×10⁵/ml). Equal amounts of cell lysates were resolved by SDS-PAGE, transferred to PVDF membrane and probed with specific antibodies against Bcl-2, Bax. Analysis was confirmed with three different sets of experiments. (C) Effect of F2 on release of cytochrome c into cytosol. Cytoplasmic and mitochondrial fractions were prepared from control and F2 treated (18.6 µg/ml for 6 h, 12 h, 24 h, 48 h) U937 cells using mitochondria/cytosol fractionation kit and cytochrome c was analyzed by Western blotting with anti cytochrome c antibody. Data shown are from one of the three experiments. β-actin served as a loading control.</p

Effect of F2 on ROS generation and mitochondrial respiration.

<p>Increase in ROS levels. U937 (2.5×10⁵/ml) cells were treated with (A) IC₅₀ concentration of F2 [18.6 µg/ml for 0–2 h (time-dependent) or (B) 0–25 µg/ml for 30 min (concentration dependent)]. After treatment, cells were washed and resuspended in PBS, incubated with CM-H₂DCFDA (5 µM) for 30 min and the fluorescence was measured using flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071672#s2" target="_blank">Materials and methods</a>. For the inhibition of ROS generation, cells were pre-treated with NAC (2.5 mM) for 3 h before treatment with F2 (18.6 µg/ml; 30 min) and ROS was similarly quantified. Values are expressed as Mean Fluorescence Intensity ± SEM of three independent experiments (**p<0.01, ***p<0.001). (C) Effect of anti-oxidant on F2 induced cytotoxicity. Cells (2.5×10⁴/100 µl of RPMI 1640 medium/well) were pre-incubated with anti-oxidant NAC (2.5 mM; 3 h) followed by treatment with F2 (0–50 µg/ml; 48 h). Cell viability was evaluated by MTT assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071672#s2" target="_blank">Materials and methods</a>. Histograms represent percentage cell viability (Mean ± SEM) obtained at the IC₅₀ concentration of F2 (18.6 µg/ml) and has been derived from at least three experiments in duplicate (***p<0.001 compared to only F2 treated cells). (D) Effect of anti-oxidant on F2 induced apoptosis. Cells were treated with F2 (18.6 µg/ml; 48 h) with or without pre-incubation using NAC (2.5 mM; 3 h). They were co-stained with Annexin-V FITC and PI followed by analysis using flow cytometry as described before. Histograms represent percentage apoptotic cells and have been derived from at least three experiments (***p<0.001 compared to control cells). (E) Inhibition of respiration. U937 cells were treated with F2 (18.6 µg/ml; 30 min) with or without pretreatment with NAC (2.5 mM; 3 h). Oxygen contents were monitored by using the Oxytherm system. Data shown are from one of the three experiments.</p

Cytotoxicity of extracts of flowers of <i>Sesbania grandiflora</i>.

<p>Log phase cells (1.25–2.5×10⁴/100 µl of RPMI 1640 medium/well) were treated with hexane, ethyl acetate, n-butanol extracts of <i>Sesbania grandiflora</i> (0–100 µg/ml) for 48 h, and cell viability was measured by MTT assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071672#s2" target="_blank">Materials and methods</a>. Each IC₅₀ (Mean ± SEM) has been derived from at least three experiments in duplicate.</p

Induction of apoptosis by F2.

<p>Control and F2 treated (18.6 µg/ml; 12 h, 24 h, 48 h) U937 cells (2.5×10⁵/ml) were co-stained with PI and Annexin-V FITC followed by analysis using flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071672#s2" target="_blank">Materials and methods</a>. The figure is a representative profile of at least three experiments.</p

Involvement of caspase-dependent and caspase-independent modes of cell death.

<p>(A) Caspase activation. Activity of caspase-3, -8, -9 was measured in control and F2 (18.6 µg/ml) treated U937 cells (2.5×10⁵/ml) using colorimetric tetrapeptide substrates. Results are expressed as mean ± SEM from three independent experiments. (B) Cleavage of PARP by F2. Cell lysates were prepared and subjected to western blot analysis to check the cleavage of PARP. Data shown are from one of the three experiments. (C) Effect of pan caspase inhibitor on F2 induced cytotoxicity. Cells (2.5×10⁴/100 µl of RPMI 1640 medium/well) were pre-incubated with pan caspase inhibitor Z-VAD-FMK (20 µM; 4 h) followed by treatment with F2 (0–100 µg/ml; 48 h). Cell viability was evaluated by MTT assay. Histograms represent percentage cell viability (Mean ± SEM) obtained at the IC₅₀ concentration of F2 (18.6 µg/ml) and has been derived from at least three experiments in duplicate. (D) Effect of pan caspase inhibitor on F2 induced apoptosis. Cells were treated with F2 (18.6 µg/ml; 48 h) with or without pre-incubation using Z-VAD-FMK (20 µM; 4 h). They were co-stained with Annexin V-FITC and PI followed by analysis using flow cytometry. Histograms represent percentage of apoptotic cells and have been derived from at least three experiments (***p<0.001 compared to control cells). (E) Translocation of AIF. Cytoplasmic and nuclear fractions were prepared from control and F2 treated U937 cells using nuclear/cytosol fractionation kit and translocation of AIF was analyzed by Western blotting. Analysis was confirmed with three different sets of extracts. Laminin served as a loading control.</p

Induction of autophagy.

<p>(A) Formation of AVO. Control and F2 (18.6 µg/ml; 0–48 h) treated U937 cells (2.5×10⁵/ml) were stained with acridine orange (1 µg/ml) for 15 min and AVO formation observed under a fluorescence microscope (60X). At least 20 microscopic fields were observed for each sample. Formation of autophagosomes. (B) Control and F2 (18.6 µg/ml; 48 h) treated U937 cells were stained with toluidine blue and photographed under a light microscope (60X). At least 20 microscopic fields were observed for each sample. (C) TEM microphotographs showing the ultrastructure of control and F2 (18.6 µg/ml; 12 h, 24 h, 48 h) treated U937 cells. Double membraned autophagosomes (black arrow) and autolysosomes (black arrow heads) were observed in treated cells. The figure is a representative profile of at least three experiments.</p