DT-GMCSF kills other AML cells by necroptosis.

<p>(A). Cell viability of Kusami cells as determined by MTS assays after 48 hours of treatment with DT-GMCSF 3-fold serial dilution (mean+/−SEM from 3 replicates). (B). DT-GMCSF (450 µg ml⁻¹) induce caspase activity in Kusami cells as determined using a caspase 3/7 luminogenic assay (mean+/−SEM from 3 replicates). (C). Cell viability of Kusami cells in response to DT-GMCSF (450 µg ml⁻¹) in the presence of zVADfmk (50 µM) and/or Necrostatin-1 (50 µM) was measured using MTS assays (mean+/−SEM from 3 replicates). (D). Cell viability of HL60 cells as determined by MTS assays after 48 hours of treatment with DT-GMCSF 3-fold serial dilution (mean+/−SEM from 3 replicates). (E). DT-GMCSF (450 µg ml⁻¹) induce caspase activity in HL60 cells as determined using a caspase 3/7 luminogenic assay (mean+/−SEM from 3 replicates). (F). Cell viability of HL60 cells in response to DT-GMCSF (450 µg ml⁻¹) in the presence of zVADfmk (50 µM) and/or Necrostatin-1 (50 µM) was measured using MTS assays (mean+/−SEM from 3 replicates). (* P<0.05).</p

Diphtheria toxin alone is sufficient to activate both cell death pathways (mean+/−SEM from 3 replicates).

<p>(A). MTS assays measuring U937 cell viability in response to Diphtheria toxin dose curve. (mean+/−SEM from 3 replicates) (B). Diphtheria toxin (150 ng ml⁻¹) activates caspases in U937 cells as determined using a caspase 3/7 specific luminogenic substrate (mean+/−SEM from 3 replicates). (C) DNA from U937 cells treated with Diphteria toxin (150 ng ml⁻¹) or TRAIL (150 ng ml⁻¹)/cycloheximide (0.5 µg/ml) were run on a 2% agarose gel to detect nucleosomal laddering as a marker of apoptosis. (D). Pretreating U937 cells with zVADfmk (50 µM) and/or Necrostatin-1 (50 µM) provides protection against Diphtheria toxin (150 ng ml⁻¹) as measured by MTS assays (mean+/−SEM from 4 replicates). (* P<0.05) (**P<0.01) (#P<0.005) (##P<0.001).</p

Inhibition of protein synthesis is sufficient to activate apoptosis and necroptosis in U937 cells.

<p>(A). Cycloheximide (5 µg ml⁻¹) induce caspase activity in U937 cells in a manner similar to both Diphtheria toxin and DT-GMCSF as determined using a caspase 3/7 luminogenic assay (mean+/−SEM from 3 replicates). (B). Cell viability of U937 cells in response to cycloheximide (5 µg ml⁻¹) in the presence of zVADfmk (50 µM) and/or Necrostatin-1 (50 µM) was measured using MTS assays (mean+/−SEM from 4 replicates). (C). MTS assays were used to measure the ability of Geldanamycin (0.5 µM) and zVADfmk (25 µM) to protect against cycloheximide (5 µg ml⁻¹) induced cell death in U937 cells (mean+/−SEM from 3 replicates) (* P<0.05) (**P<0.01) (#P<0.005) (##P<0.001).</p

Survival of Sprague Dawley rats (250 to 300 g) treated with single intravenous bolus infusion of LeTx (0.03 mg PA+0.015 mg LF) or EdTx (0.15 mg PA+0.075 mg EF) and monitored for two weeks.

<p>These are the same doses used for telemetry and ECHO experiments. N = 12 for each experiment. Kaplan-Meier graphs prepared.</p

Mortality of Anthrax Toxins^*

*<p>n = the number of animals used in each dose study</p

Boxplots at different times post anthrax toxin bolus iv infusion of echocardiographic parameters:

<p>A. LeTx treated rats at zero, one and two hr post-infusion, velocity of propagation was 24±5, 34±7 and 46±17 cm/sec, respectively, with significant difference (P = 0.05 at one hr compared to controls and P = 0.007 at two hr compared to controls); B. LeTx treated rats at zero and one hr post-infusion, left ventricular diastolic area was 0.98±0.07 and 1.15±0.06, respectively, with significant difference (P = 0.01); C. LeTx treated rats at zero and one hr post-infusion, left ventricular systolic area was 0.79±0.08 and 0.98±0.10 cm, respectively, with significant difference (P = 0.02); D. EdTx treated rats at zero, one and two hr post-infusion, heart rate was 326±49, 371±28, and 393±10 beats per min, respectively, with significant difference (P = 0.05 at one hr and P = 0.001 at two hr compared to controls). The box represents the middle 50% of the data. The line through the box represents the median. The line (whiskers) extending from the box represent the upper and lower 25% of the data. The line of each plot connects the means of the sample.</p

Serum levels of PA after single bolus intravenous infusions of different doses of LeTx to Sprague Dawley rats (250 to 300 g).

<p>Concentrations of toxin components measured by ELISA as described in the text. Peak concentrations and half-lives shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000466#pone-0000466-t002" target="_blank">Table 2</a>.</p

Anthrax toxins in serum samples^*

*<p>Three animals were used in each dose study; ND, not determined.</p