EGCG induces LMP.

<p>(A) Effect of EGCG on intracellular acidic compartments. HepG2 cells were with EGCG (60 µM) for indicated durations or with Baf A1 (50 nM) for 6 h. The acidic compartments were labeled by 5 nM Lyo-Tracker Red and examined by confocal (scale bar: 20 µm). (B) EGCG induces the leakage of cathepsins from lysosome to cytosol. Cell fractionation was performed to separate lysosomal and cytosolic fractions in HepG2 treated with 60 µM EGCG in serum-free medium as indicated. Cathepsin D was detected by western blotting in the different fractions and whole cell lysates. LAMP-1 was used as a marker for lysosome. (C) EGCG causes lysosomal neutralization and cytosolic acidification. HepG2 cells were treated with 60 µM EGCG as indicated in serum-free medium, followed by staining with 5 µg/ml acridine orange (AO) for 30 min and analyzed by confocal (scale bar: 20 µm).</p

ROS mediates LMP and cell death induced by EGCG.

<p>(A) EGCG induces intracellular ROS production. HepG2 cells were treated with EGCG (60 µM) for 6 h in full medium or in serum-free medium Treatment with H₂O₂ (200 µM) for 3 h was used as a positive control. The intracellular ROS was detected by CM-H₂DCFDA and analyzed under a fluorescence microscope. (B) NAC prevents ROS formation induced by EGCG in serum-free medium. Cells were treated with EGCG (60 µM×6 h) in the absence or presence of N-acetylcysteine (NAC, 5 mM). (C) Protection by NAC of EGCG-induced cell death. HepG2 cells were treated by EGCG (60 µM) or H₂O₂ (200 µM) as shown for 12 h in the absence or presence of 5 mM NAC. The cell viability was determined by Hoechst-PI double staining (n = 3, mean ± SD). **<i>P</i><0.005 in comparison to the group without NAC (Student's <i>t</i>-test). (D) NAC prevents cathepsin D translocation caused by EGCG. HepG2 cells were treated with EGCG (60 µM×6 h) with or without 5 mM NAC. Both the lysosomal and cytosolic fractions were analysed by western blot. (E) NAC prevents EGCG-induced cytosolic acidification. HepG2 cells were treated with EGCG (60 µM×6 h) or H₂O₂ (200 µM×3 h) with or without 5 mM NAC. Cells were then stained with AO for 30 min and analyzed by confocal (scale bar: 20 µm). (F) CQ fails to affect ROS production induced by EGCG. HepG2 cells were cultured in serum-free medium for 1 h, then added 60 µM EGCG, with or without the presence of 20 µM CQ for 6 h. (G) Illustration for the mechanisms underlying EGCG-mediated caspase-independent cell death, involving ROS and LMP.</p

Serum starvation enhances EGCG-induced cell death independent of caspase.

<p>(A) Serum deprivation promotes EGCG-induced cell death in a concentration-dependent and time-course manner. HepG2 cells were treated with different doses of EGCG in full or serum-free medium for 12 h (left panel) or with 60 µM EGCG for different time as indicated (right panel). The cell viability was determined by Hoechst-PI double staining (n = 3, mean ± SD). (B) Representative pictures of Hoechst-PI double staining. HepG2 cells were cultured in full medium (as a control); treated with 60 µM EGCG for 12 h in serum-free medium; or incubated with 20 ng/ml TNFα and 10 µg/ml CHX for 12 h in full medium (as a positive control for apoptosis). (C) EGCG induces caspase-independent cell death. HepG2 cells were treated with EGCG (60 µM×24 h) or in the absence or presence of 40 µM z-VAD-fmk. The co-treatment with TNFα (20 ng/ml) and CHX (10 µg/ml) for 12 h was used as a positive control. Cell viability was determined as described in Panel A. **<i>p</i><0.005 comparing to the group without z-VAD (Student's <i>t</i>-test, n = 3). (D) No caspase-3 activation and PARP cleavage cause by EGCG-induced cell death. Cells were treated with EGCG or TNF/CHX as described in panel C, and cell lysates were collected and subject to western blot.</p

EGCG induces cytosolic vacuolization.

<p>(A) Morphological alterations of EGCG-treated cells in serum-free medium were analyzed using light microscopy. Representative pictures of HepG2 cell treated with EGCG at indicated concentrations for 12 h (upper panel) or with 60 µM EGCG for indicated time courses (lower panel) are shown (scale bar: 50 µm). (B) HepG2 cells were treated with EGCG (60 or 240 µM) for 12 h. The cells were then fixed and stained by hematoxylin, then analyzed by light microscopy (scale bar: 30 µm). (C) The vacuole contents are not lipid droplets. HepG2 cells were EGCG (60 µM) for 12 h. Cultured cells in full medium for 12 h were used as a negative control, and cells treated with 1 mM oleate acid (OA) in DMEM medium containing 1%BSA for 12 h were used as the positive control. The cells were fixed and stained with 0.5% Oil Red O and hematoxylin (scale bar: 30 µm). (D) The vacuoles are of lysosome origin. Immunofluorescence staining of LAMP-1 was performed in MEF cells after treatment with or without EGCG (60 µM) for 9 h (scale bar: 10 µm).</p

EGCG blocks autophagic flux.

<p>(A) EGCG increases LC3-II and p62 protein level. HepG2 and MEF cells were treated with EGCG (60 µM) for indicated durations in full medium or in serum-free medium as indicated. Cell lysates were collected for western blot. (B) EGCG increases the formation of GFP-LC3 puncta. MEF with stable expression of GFP-LC3 (m5–7 cells) were treated with or without 60 µM EGCG for 9 h in serum-free medium and analyzed by confocal microscopy (scale bar: 20 µm). (C) EGCG does not promote autohpagic flux. HepG2 cells were treated in serum-free medium with 60 µM EGCG, 50 nM Baf A1, or both for 9 hours. Cell lysates were collected and subject to western blot. (D) EGCG has no effect on autophagosome-lysosome fusion. MEF cells with stable expression of GFP-LC3 were cultured in serum-free medium for 9 h (as a control); treated with EGCG (60 µM×9 h) in serum-free medium; or cultured in EBSS medium for 2 h (as a positive control). Cells were then, stained LAPM-1 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046749#pone-0046749-g002" target="_blank">Figure 2D</a>. Cells were analyzed by confocal microscopy (scale bar: 10 µm).</p

EGCG induces cell death via LMP.

<p>(A) Different lysosome inhibitors present different effects on EGCG-induced cell death. HepG2 cells were treated for EGCG (60 µM×24 h) in serum-free medium in the absence or presence of various inhibitors, including E64-D (10 µg/ml) + pepstatin A (10 µg/ml), chloroquine (CQ, 25 µM), Baf A1 (50 nM). The cell viability was determined by Hoechst-PI double staining (n = 3, mean ± SD). The <i>p</i> values were determined using Student's <i>t</i>-test (*<i>P</i><0.05, **<i>P</i><0.005). (B) CQ, but not Baf A1 blocks the cytosolic acidification induced by EGCG. HepG2 cells were with EGCG (60 µM), CQ (25 µM) or Baf A1 (50 nM) in serum-free medium for 6 h followed by staining with 5 µg/ml AO for 30 min and analyzed by confocal (scale bar: 20 µm).</p

EGCG causes cell death independent of autophagy.

<p>(A) MEF cells with inducible deletion of Atg5 (m5–7 cells) were cultured with or without 10 ng/ml Dox for 4 days, then EGCG (60 µM×6 h), and cell lysates were collected and subject to western blot. (B) The formation of vacuoles is Atg5-independent. m5–7 cells as described in panel A were treated with EGCG (60 µM) for 12 h and observed under light microscopy (scale bar: 30 µm). (C) EGCG-induced cell death is independent of autophagy. m5–7 cells as described in panel A were treated with EGCG (60 µM) for 24 h. The cell viability was determined by Hoechst-PI double staining (n = 3, mean ± SD).</p