NAC reverses cytotoxicity and pro-caspase activity induced by combined EGCG/As treatment.

<p>(A) BAEC were pretreated with various doses (0, 1, 5, or 10 mM) of NAC for 3 h prior to EGCG/As treatment for 24 h. (B-D) In separate experiments, EC were pretreated with the indicated dose (5 mM) of NAC. (E) In flow cytometric analysis, BAEC were pretreated with 20 μM Boc-D-FMK for 3 h prior to EGCG/As treatment for 12 h. (A) Cell viability, (B) caspase activity, (C) DAPI staining, (D) Bax translocation into the mitochondria, and (E) flow cytometric analyses were performed as described in the legend of Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g002" target="_blank">2</a>. All bar graphs represent the mean ± S.D. of 3 independent experiments. Statistical analysis was accomplished as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g001" target="_blank">Fig 1</a>.</p

Combined EGCG/As treatment increases ROS generation and decreases the activity of catalase but not SOD.

<p>(A) ROS levels were determined by flow cytometric analysis using DCFH-DA staining. Cells were treated with EGCG, As, or EGCG/As (each 20 μM) for 3 h and then stained with DCFH-DA. Stained cells were analyzed by flow cytometry using Cellquest software. The data are representative of 3 independent experiments. (B) SOD activity was assessed in cells treated with EGCG, As, or EGCG/As (each 20 μM) for 30 min. (C) The catalase activity was measured in EC treated with EGCG, As, or EGCG/As (each 20 μM) for 2.5 h. (D) Lipid peroxidation was estimated by measuring the production of malondialdehyde (MDA) using the Colorimetric Microplate Assay for Lipid Peroxidation Kit (Oxford) according to the manufacturer’s protocol. All bar graphs represent the mean ± S.D. of 3 independent experiments. Statistical analysis was accomplished as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g001" target="_blank">Fig 1</a>.</p

Combined EGCG/As decreases the viability of two types of EC, HUVEC and HBMEC.

<p>(A) HUVEC were prepared and treated with various doses (0, 10, 20, 30, 40, 50 or 100 μM) of As or EGCG for 24 h. (B) In separate experiments, HUVEC were also treated with 50 μM EGCG, 10 μM As, or the combination of 50 μM EGCG and 10 μM As (EGCG/As) for 24 h. In some experiments, cells were pretreated with 5 mM NAC, 50 U/ml catalase or 1 μM SP600125 for 30 min prior to exposed to EGCG/As. (C) HBMEC were prepared and treated with various doses (0, 10, 20, 30, 40, 50 or 100 μM) of As or EGCG for 24 h. (D) In separate experiments, cells were also treated with 50 M EGCG, 50 μM As, or the combination of 50 μM EGCG and As each (EGCG/As) for 24 h. In separate experiments, cells were pretreated with 5 mM NAC, 50 U/ml catalase or 10 μM SP600125 for 30 min prior to exposed to EGCG/As. Cell viability was measured using MTT assay. All bar graphs represent the mean ± S.D. of 3–5 independent experiments. Statistical analysis was accomplished as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g001" target="_blank">Fig 1</a>.</p

JNK mediates catalase activity, ROS production, and apoptosis altered by combined EGCG/As treatment.

<p>(A) BAEC were treated with EGCG/As for the indicated times (0, 0.5, 1, 2, or 3 h). (B) After pretreatment with catalase (50 U/ml) or the JNK inhibitor SP600125 (1 μM) for 30 min, EC were treated with EGCG/As for 1 h. The level of phosphorylated JNK (p-JNK) and total JNK protein was detected by Western blot analysis. (C) Cells were prepared and stained as described in the legends of Fig 5A and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g002" target="_blank">Fig 2</a>. In some experiments, cells were pretreated as described in the legend of Fig 5B, followed by treatment with EGCG/As for 3 h. Flow cytometric analysis was performed as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g002" target="_blank">Fig 2</a>. (D) Cell viability was determined as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g001" target="_blank">Fig 1</a> using BAEC pretreated with SP600125 prior to EGCG/As treatment for 24 h. (E) Catalase activity was measured as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g002" target="_blank">Fig 2</a> using BAEC pretreated with SP600125 prior to EGCG/As treatment for 2.5 h. (F, G) Cells were prepared, and pretreated with SP600125 (F) or MG132 (20 μM) (G) for 30 min prior to treatment of EGCG/As for 2.5 h. Cell lysate (30 μg) was subjected on 10% SDS-PAGE, and the level of catalase protein was then detected as described in <b>Materials and methods</b>. All bar graphs represent the mean±S.D. of 3 independent experiments. Statistical analysis was accomplished as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g001" target="_blank">Fig 1</a>.</p

Combined EGCG/As treatment increases apoptosis in BAEC.

<p>(A) BAEC were treated with various doses (0, 5, 10, 20, 30, or 40 μM) of As or EGCG for 24 h. (B) In some experiments, cells were also treated for 24 h with 20 μM EGCG, 20 μM As, or the combination of 20 μM EGCG and As each (EGCG/As). (A, B) Cell viability was measured using MTT assay. (C, D) Cells treated with EGCG, As, or EGCG/As for 12 h. (C) Apoptotic cells were detected by DAPI staining. (D) Cells were lysed in RIPA buffer. An equal amount (20 μg) of each cell lysate was subjected to Western blot analysis. Levels of cleaved PARP expression were detected with an anti-cleaved PARP antibody. Quantifications were performed using densitometry (Image J software) and results were normalized to β-actin. (E-G) The activity of caspases (3, 8, and 9) was measured in cells treated with EGCG, As or EGCG/As for the specified times (0, 6, 12, 18, or 24 h). All line graphs represent the relative caspase activity of the control. (H) Assay for Bax translocation into the mitochondria. Cells treated with EGCG, As, or EGCG/As for 12 h were stained with FITC-conjugated anti-Bax antibody, Mitotracker as a marker of mitochondria, or DAPI. All bar graphs represent the mean ± S.D. of 3 independent experiments. The different characters refer to significant differences (<i>P <</i> 0.05) among groups, which were determined by one-way ANOVA followed by post hoc Student-Newman-Keuls analysis.</p

Catalase reverses cytotoxicity and pro-caspase activity induced by combined EGCG/As treatment.

<p>BAEC pretreated with catalase (50 U/ml) for 30 min were exposed to EGCG/As for 24 h. (A) Cell viability, (B) Western blot analysis using the indicated antibodies, and (C) Bax translocation into the mitochondria were determined as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g001" target="_blank">Fig 1</a>. All bar graphs represent the mean ± S.D. of 3 independent experiments. Statistical analysis was accomplished as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g001" target="_blank">Fig 1</a>.</p