Comparison of chemical structures.

<p>Structures of (<b>A</b>) Pancratistatin (PST) and (<b>B</b>) JC-TH-acetate-4 (JCTH-4).</p

JCTH-4 alone and in combination with CC yields apoptotic morphology in OS cells.

<p>Nuclear and cellular morphology of (<b>A</b>) Saos-2 cells after 96 hours of treatment and (<b>B</b>) U-2 OS cells after 72 hours of treatment. Cells were treated with JCTH-4, CC, and solvent control (Me₂SO). Post treatment, the cells were stained with Hoechst 33342 dye. Corresponding phase micrographs are shown below the Hoechst micrographs. Apoptotic morphology is evident in cells with bright and condensed nuclei accompanied by apoptotic bodies, as well as cell shrinkage and blebbing. Images were taken at 400× magnification on a fluorescent microscope. Scale bar = 15 µm. All images are representative of 3 independent experiments.</p

JCTH-4 and CC do not yield apoptotic morphology in HOb and NFF cells.

<p>Nuclear and cellular morphology of (<b>A</b>) HOb and (<b>B</b>) NFF cells after 72 hours of treatment. Cells were treated with JCTH-4, CC, and solvent control (Me₂SO). Post treatment, the cells were stained with Hoechst 33342 dye. Corresponding phase micrographs are shown below the Hoechst micrographs. Apoptotic morphology is evident in cells with bright and condensed nuclei accompanied by apoptotic bodies, as well as cell shrinkage and blebbing. Images were taken at 400× magnification on a fluorescent microscope. Scale bar = 15 µm. All images are representative of 3 independent experiments.</p

JCTH-4 and CC do not induce autophagy in Hob and NFF cells.

<p>MDC staining was used to detect the presence of autophagic vacuoles in (<b>A</b>) HOb and (<b>B</b>) NFF cells after 72 hours of treatment with JCTH-4, CC, and solvent control (Me₂SO) at the indicated concentrations. Bright blue punctate marks are indicative of autophagic vacuoles. Corresponding phase and PI micrographs are shown below the MDC images. Scale bar = 15 µm. All images are representative of 3 independent experiments.</p

JCTH-4 causes selective cytotoxicity in OS cells in a time and dose-dependent manner.

<p>Effect of JCTH-4 on cellular viability of OS cells was determined by the WST-1 based colorimetric assay. (<b>A</b>) Saos-2 and (<b>B</b>) U-2 OS cells were treated with JCTH-4 and the WST-1 reagent was used to quantify cell viability. Absorbance was read at 450 nm and expressed as a percent of the control (Me₂SO). Values are expressed as mean ± SD from quadruplicates of 3 independent experiments. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 versus solvent control (Me₂SO). (<b>C</b>) Effect on cellular viability of HOb and NFF cells treated with JCTH-4 compared to Saos-2 and U-2 OS cells after 72 hours. The WST-1 reagent was used to quantify cellular viability. Absorbance was read at 450 nm and expressed as a percent of the solvent control (Me₂SO). Values are expressed as mean ± SD from quadruplicates of 3 independent experiments. *<i>p</i><0.005 versus Saos-2 cells; #<i>p</i><0.005 versus U-2 OS cells.</p

CC potentiates the cytotoxicity of JCTH-4 selectively in OS cells.

<p>Effect of JCTH-4 & CC in combination on cellular viability of OS cells was determined by the WST-1 based colorimetric assay. (<b>A</b>) Saos-2 (96 hours), (<b>B</b>) U-2 OS (72 hours), (<b>C</b>) HOb (72 hours), and (<b>D</b>) NFF (72 hours) cells were treated with JCTH-4 and CC and the WST-1 reagent was used to quantify cellular viability. Absorbance was read at 450 nm and expressed as a percent of the solvent control (Me₂SO). Values are expressed as mean ± SD from quadruplicates of 3 independent experiments. *<i>p</i><0.05, **<i>p</i><0.01, versus solvent control (Me₂SO); †<i>p</i><0.001 versus 0.25 µM JCTH-4; ††<i>p</i><0.01 versus 0.5 µM JCTH-4; #<i>p</i><0.001 versus 5 µM CC; @<i>p</i><0.001 versus 0.25 µM JCTH-4+5 µM CC treatment with Saos-2 cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028780#pone-0028780-g003" target="_blank">Figure 3A</a>); &<i>p</i><0.01 versus 0.5 µM JCTH-4+5 µM CC treatment with U-2 OS cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028780#pone-0028780-g003" target="_blank">Figure 3B</a>).</p

JCTH-4 dissipates MMP alone and in combination with CC in OS cells.

<p>Effect of JCTH-4 and CC on MMP in (<b>A</b>) Saos-2 cells after 96 hours of treatment and (<b>B</b>) U-2 OS cells after 72 hours of treatment was examined by TMRM staining. Cells were grown on coverslips, treated with the indicated concentrations of JCTH-4, CC, and solvent control (Me₂SO) and stained with TMRM and Hoechst dye. Images were taken at 400× magnification on a fluorescent microscope. Red fluorescent punctuate marks are indicative of mitochondria with intact MMP. Scale bar = 15 µm. All images are representative of 3 independent experiments.</p

JCTH-4 directly causes mitochondrial ROS production and release of apoptogenic factors independent of caspases.

<p>(<b>A</b>) Saos-2 and (<b>B</b>) U-2 OS isolated mitochondria were treated directly with JCTH-4, CC, PQ, and solvent control (Me₂SO), and incubated with Amplex Red and horseradish peroxidase for 2 hours. Subsequently, fluorescence readings were taken at Ex. 560 nm and Em. 590 nm and expressed as relative fluorescence units (RFU). Statistics were performed using GraphPad Prism version 5.0. Image is representative of 3 independent experiments demonstrating similar trends. Values are expressed as mean ± SD of quadruplicates of 1 independent experiment. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 versus solvent control (Me₂SO); †<i>p</i><0.01 versus 0.25 µM JCTH-4; @<i>p</i><0.01 versus 0.5 µM JCTH-4; #<i>p</i><0.01 versus 5 µM CC. Isolated mitochondria samples treated directly with JCTH-4, CC, and solvent control (Me₂SO) for 2 hours were also centrifuged, producing mitochondrial pellets and post mitochondrial supernatants which were examined for retention and release of apoptogenic factors respectively via western blot analyses; (<b>C</b>) Retention of AIF and release of EndoG by U-2 OS cell mitochondria and (<b>D</b>) release of AIF by Saos-2 cell mitochondria was monitored. Mitochondrial pellets were probed for SDHA to serve as loading controls. Densitometric analyses were performed using ImageJ software and statistics were calculated using GraphPad Prism version 5.0. Image is representative of 3 independent experiments demonstrating similar trends. Values are expressed as mean ± SD of triplicates of one independent experiment. *<i>p</i><0.01, **<i>p</i><0.001 versus solvent control (Me₂SO); †<i>p</i><0.01 versus 0.25 µM JCTH-4; #<i>p</i><0.01 versus 5 µM CC. (<b>E</b>) Saos-2 cells were treated with broad spectrum caspase inhibitor Z-VAD-FMK with and without JCTH-4 for 72 hours. WST-1 reagent was used to quantify cell viability. Absorbance was read at 450 nm and expressed as a percent of solvent control (Me₂SO). Values are expressed as mean ± SD from quadruplicates of 3 independent experiments. *<i>p</i><0.001 versus solvent control (Me₂SO); ns = not significant.</p

PLX is Well-Tolerated in Mice Models.

<p>Balb/C mice were divided into three groups (3 animals/control (untreated), 3 animals/gavage control (vehicle treatment) and 4 animals/treatment group). The control untreated group was given plain filtered water, while the second and third group was given 50 mg/kg/day vehicle (DMSO) or PLX, respectively. Mice were assessed for toxicity with protein urinalysis by Bradford Assay and dipstick analysis (A) and weight changes (B). (C) Hematoxylin and Eosin stained tissue sections of the liver, heart and kidney of control versus PLX treated group. Images were obtained on a bright field microscope at 63× objective.</p

Quantification of Cell Death Induction Following PLX Treatment.

<p>Image-Based cytometry was used to quantify apoptotic induction (% Annexin V positive), followed by necrosis (% PI positive) in E6-1 and HT-29 cells following PLX treatment. The lack of annexin V or PI staining was used as an indication of live cells following treatment (%Annexin V/PI negative cells) (*P<0.05, ** P<0.003, ***P<0.0001). (E) To further confirm the induction of apoptosis.</p