The integrity of the cell membrane is needed for the function of avicin D.

<p>Proliferating Jurkat (A, C) and Daudi (B, D) cells were pretreated with methyl-β-cyclodextrin (MCD) for 8 h. After washing away the MCD, cells were exposed to 2 µg/ml Avicin D for 24 h in DMEM medium with or without 10% FCS. Cells were fixed and stained with PI. Flow cytometry analysis was carried out to detect sub-G1 (apoptotic cells) and cell cycle distribution. A. Percentage of sub-G1 Jurkat cells after the treatment. Data shown are means±S.D. of three independent experiments. B. Percentage of sub-G1 Daudi cells after the treatment. Data shown are means±S.D. of three independent experiments. C. Representative FCM graphs of Jurkat cells after the treatment. D. Representative FCM graphs of Daudi cells after the treatment.</p

Avicin D-induced apoptosis is lipid raft-dependent.

<p>(A & B) Proliferating Jurkat cells were pretreated with 2.5 mg/ml of MCD for 1 h, followed by 2 µg/ml of avicin D for 24 h. A. Cell death was quantified using a cell death ELISA showing enrichment of nucleosomes in the cytoplasmic fraction of Jurkat cells. Values represent the mean±S.D. (n = 3). B. Cell viability was determined by trypan blue exclusion assay. The number of surviving cells was counted as a percentage of total cells. C. MCD inhibits the activity of avicin D in a dose-dependent manner. Increasing concentrations of MCD were applied to Jurkat cells. One hour after treatment with MCD, 2 µg/ml of avicin D were added to the medium. Cell viability was determined by trypan blue exclusion assay after 24 h. Jurkat cells without treatment with MCD and avicin D were set as 100%. D. Jurkat cells were pretreated with 2.5 mg/ml MCD for 1h, followed by 2 µg/ml avicin D for 8 h. The cells were then fixed and stained with FITC-CTxB subunits to identify rafts (green fluorescence) and with anti-Fas antibody to identify Fas (red fluorescence). Area of colocalization between membrane rafts and Fas in the merge panels is yellow.</p

Aberrations in Fas and its downstream signaling molecules abrogate the effects of avicin D.

<p>Parental Jurkat cells and Fas-, FADD-, Caspase-8-, and RIP-deficient Jurkat cells were treated with 2 µg/ml of Avicin D for 24 h. A. Detection of apoptotic molecules by Western blot. 50 ìg of total protein were loaded in each lane. β-Actin was detected to serve as an internal control. B. The percentage of apoptotic cells. After the treatment, the cells were fixed and stained with PI. Flow cytometry (FCM) analysis was performed to detect sub-G1 (apoptotic cells). C. Representative FCM graphs of parental, Fas-deficient, and RIP-deficient Jurkat cells.</p

Avicin D activates Caspase-8–mediated cell death.

<p>Proliferating Jurkat cells were exposed to different doses of avicin D for 24–72 h. A. Cell death was measured by trypan blue exclusion assay. The number of dead cells was counted as a percentage of total cells. B. Cell viability was measured by a CellTiter-Glo Luminescent Cell Viability Assay kit. Data are shown as mean±S.D. (n = 5). The number of surviving cells was counted as a percentage of total cells. C and D. Cell death and cell viability were measured in NB4 cells exposed to 0–4 µg/ml of avicin D for 24–48 h as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008532#s2" target="_blank">Methods</a>. Data are shown as mean±S.D. (n = 3). The number of dead or surviving cells was counted as a percentage of total cells. E. Proliferating Jurkat cells were exposed to 2 µg/ml of avicin D for 24 h. Cleavage of Caspase-8 induced by avicin D was analyzed by Western blot. 50 µg of total protein were loaded in each lane. α-Tubulin was detected to serve as an internal control. F. Time-dependent activation of Caspase-8 and apoptotic regulators in avicin D-treated Jurkat and NB4 cells. G. Time-dependent activation of Caspase-8 and apoptotic regulators in avicin D-treated U2OS cells.</p

Avicin D recruits Fas and its downstream signaling molecules into lipid rafts.

<p>Proliferating Jurkat cells were pretreated with or without 2.5 mg/ml MCD for 1 h, followed by 2 µg/ml Avicin D for 24 h. After the treatment, the cells were lysed in 1% Triton X-100 and subjected to discontinuous sucrose density gradient centrifugation. Components in each fraction were analyzed by Western blot. Location of lipid rafts was determined by a dot blot using CTxB subunits conjugated to horseradish peroxidase. A. Components of lipid rafts in control and avicin D-treated Jurkat cells. B. Components of lipid rafts in MCD with/without avicin D-treated Jurkat cells.</p

Avicin D induces clustering of Fas in lipid rafts.

<p>Jurkat cells were treated with 2 µg/ml of avicin D for 0–8 h. After the treatment, the cells were fixed and stained with FITC-CTxB subunits to identify lipid rafts (green fluorescence) and with anti-Fas antibodies to identify Fas (red fluorescence). Area of colocalization between membrane rafts and Fas in the merge panels is yellow.</p

Effect of avicin D on GRE-dependent gene expression.

<p>(A) A549 cells transfected with p (GRE)₂-50huIL6P-luc+ were treated with avicin D (1 µM) or Dex (1 µM) for 2–16 hrs. Luciferase activity was measured in cell lysates, and normalized for TK renilla luminescence as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. (B) Effect of avicin D and RU486 on Dex-induced luciferase activity. A549 cells transfected with p (GRE)₂-50huIL6P-luc+ were pre-treated with avicin D (1 µM) or RU486 (1 µM) for 2 hrs, prior to being exposed to Dex (1 µM) or avicin D (1 µM) for 16 hrs. Luciferase activity was measured in cell lysates, and normalized for TK renilla luminescence as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. Luciferase activity has been expressed as relative luciferase units (RLU). (C) A549 cells were treated with Avicin D (1 µM) for 0–120 min or with Dex (1 µM) for 60 min. Western blot analysis of the nuclear extracts was performed using anti-phospho GR (Ser211) antibodies. Actin levels have been shown as a loading control.</p

A model of avicin D-GR interaction.

<p>The Avicin-D warhead was docked into the crystal structure of RU-486 bound to the antagonist form of glucocorticoid (pdb code: 1NHZ) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. (A) Distances between the Sg of three cystein residues and the olefin groups of Avicin-D warhead (green carbon atoms). (B) Ribbon structure of 1NHZ with RU-486 (brown carbon atoms) and the model of the Avicin-D warhead (green carbon atoms). (C) Same as (B) except focused on the binding pocket of RU-486.</p

Role of different GR domains in avicin D-mediated inhibition of NF-κB activity.

<p>HEK 293T cells were transiently transfected with mock DNA, or plasmids carrying either wild type GRα or deletion variants of GR. After transfection, cells were pre-treated with Avicin D (1 µM) for 2 hrs, followed by a 4 hrs treatment with TNF (1 nM). Luciferase activity was measured in the cell lysates, and normalized for TK renilla luminescence as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. Luciferase activity has been expressed as % change over the untreated control, which in turn is taken as 100%.</p

Avicin D inhibits activation of NF-κB.

<p>(A) A549 cells were pre-treated with 1 µM each of either avicin D or Dex for 24 hrs. Following the pre-treatment, cells were exposed to TNF (1 nM) for another 24 hrs. At the end of the TNF treatment, cell supernatants were collected. IL-6 levels were measured using an ELISA kit. (B) A549 cells transfected with p (IL6κB)3-50huIL6P-luc+ were either untreated or pre-treated with Avicin D/Dex (1 µM each) for 2 hrs, followed by a 4 hrs treatment with TNF (1 nM). Cell lysates were assayed for luciferase activity, and normalized for TK renilla luminescence as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. Luciferase activity has been expressed as relative luciferase units (RLU). (C) Normal and GR over expressing HEK 293T cells were transfected with 100 ng of p (IL6κB)3-50huIL6P-luc+ as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. Cells were next pre-treated treated with Avicin D/Dex (1 µM each) and treated with TNF (1 nM) as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#pone-0028037-g006" target="_blank">Fig. 6B</a>. Luciferase activity was measured in cell lysates, and normalized for TK renilla luminescence as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. Luciferase activity has been expressed as % change over the untreated control, which in turn is taken as 100%. (D) A549 cells transfected with p(IL6κB)3-50huIL6P-luc+ were pre-treated with avicin D/Dex (1 µM each) either as single agents or in combinations for 2 hrs, followed by a 4 hrs treatment with TNF (1 nM). Cell lysates were assayed for luciferase activity and normalized for TK renilla luminescence as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028037#s2" target="_blank">methods</a>. Luciferase activity has been expressed as relative luciferase units (RLU).</p