Comparison of cytotoxic effects and immunotoxin specificity in Tac-receptor expressing cells.

<p>Tac-TGN38 and Tac-furin cells were treated with up to 10 ng/ml of LMB-2. After a 24 hour incubation with immunotoxin, cells were washed and the cells remaining in the well were used for <b>A</b>) Hoechst-based cell count assay or <b>B</b>) LDH assay as described in Methods. Panels A and B show % cytotoxicity plotted against the concentration of LMB-2. The data points represent the cytotoxicity values averaged over several experiments, cell count assay (n = 9), LDH assay (n = 8). The standard error of the mean is shown. <b>C</b>) Tac-TGN38 and Tac-furin cells were seeded and then treated with up to 10 ng/ml of LMB-2 or Erb38 one day after plating. After a 24 hour incubation with immunotoxin, cytotoxicity was assessed by a cell count assay. The number of cells remaining after toxin treatment is shown as a fraction of cells incubated without toxin (n = 5). The standard error of the mean is shown.</p

Effect of lysosomal protease inhibitors on LMB-2 cytotoxicity.

<p>Tac-furin cells were pretreated with 100 µM Leupeptin (Leu), E-64, Chymostatin (Chy), a combination of all three inhibitors or DMSO control for 24 hours. Cells were subsequently treated with 1 ng/ml LMB-2 in the continued presence of inhibitors. After a 24 hour incubation with immunotoxin, cells were counted using the Hoechst-based assay. The number of cells remaining after toxin treatment is shown as a fraction of cells incubated without toxin normalized to the DMSO control (n = 6 wells). The standard error of the mean is shown. Similar experiments were performed twice and one representative data set is shown.</p

Analysis of LMB-2 processing and degradation.

<p><b>A</b>) Tac-TGN38 and Tac-furin cells were seeded, and one day after plating the cells were incubated with 1 µg/ml of LMB-2 for 5, 15, 30, 60 or 120 minutes or left untreated. At each time point cells were lysed and proteins were analyzed by immunoblot with anti-<i>Pseudomonas</i> exotoxin A antibody and anti-tubulin antibodies. Proteins were detected using enhanced chemiluminescence reagents. This experiment was replicated 2 times with similar results, and one set of representative blots are shown. Tac-TGN38 (<b>B</b>) and Tac-furin (<b>C</b>) cells were incubated for 5 minutes with 750 ng/ml LMB-2 and then chased in immunotoxin-free medium for up to 270 minutes. At each time point cells were lysed in SDS. Samples were adjusted so that the amount of full length LMB-2 was the same at time 0 for each cell line. Subsequent analysis was by immunoblot using an antibody for <i>Pseudomonas</i> exotoxin A. LMB-2, tubulin and molecular weight standards are indicated. Individual bands were quantified using Metamorph. The right panels show the normalized band intensities at different chase times averaged over 4 experiments +/− standard error of the mean. The kinetic data for Tac-TGN38 were fit using SigmaPlot based on a single three parameter equation for exponential decay y  =  y₀ + ae^−bt, and Tac-furin data were fit using four parameter equation for exponential decay with the equation y  =  ae^−bt+ce^−dt (where a+c  = 100). Graph insets show the calculated t_1/2 values for each cell line.</p

The data in Figure 2 were fit in SigmaPlot using a four parameter logistic model (Hill-Slope model) with the equation y = y₀+(a/(1+(x/x₀)^b)) where b = slope, y = concentration, x₀ = EC₅₀ and a = maximum response [26], [27], [28], [38].

<p>The p-value for EC₅₀ when calculated for Cell count assay was 0.0003, and for LDH assay was <0.0001. The p-values for Maximum % killing were <0.0001 for both cell count and LDH assays.</p

Comparison of cell toxicity at comparable levels of immunotoxin binding.

<p>Tac-TGN38 and Tac-furin cells were treated with the indicated dose of LMB-2. After a 24 hour incubation with immunotoxin, cells were washed, and the cells remaining in the well were used for <b>A</b>) Hoechst-based cell count assay or <b>B</b>) LDH assay as described in Methods. Panels A and B show the relative cytotoxicity plotted against the amount of LMB-2 adjusted to reflect the different Tac expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047320#pone-0047320-g003" target="_blank">Figure 3C</a>) in the cell lines ([LMB-2] × Tac expression). Here the cytotoxicity has been normalized so that a value of 1 represents the maximum killing observed with the highest dose of LMB-2 for each cell line (relative cytotoxicity). The data points represent the cytotoxicity values averaged over several experiments, cell count assay (n = 9), LDH assay (n = 8). The standard error of the mean is shown.</p

t_1/2 for degradation were based on exponential decay curve fitting equation (see figure 5 legend for equation).

<p>The EC₅₀'s were also calculated after correcting for Tac expression and fitted in SigmaPlot using a four parameter logistic model (Hill-Slope model) with the equation y = y₀+(a/(1+(x/x₀)^b)) where b = slope, y = concentration, x₀ = EC₅₀ and a = maximum response. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047320#pone.0047320-Dudley1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047320#pone.0047320-Khinkis1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047320#pone.0047320-LaurenceMLevasseur1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047320#pone.0047320-NIH1" target="_blank">[38]</a>. The p-values for Tac-TGN38: Tac-furin were 0.03.</p

Transport pathways taken by Tac chimeras.

<p>Tac-furin (<b>A</b>) and Tac-TGN38 (<b>B</b>) are internalized from the plasma membrane (PM) and transported to the sorting endosome (SE). From here, Tac-TGN38 passes through the endocytic recycling compartment (ERC). Most of the Tac-TGN38 returns to the PM, but about 20% of the molecules in the endocytic recycling compartment are trafficked to the trans Golgi network (TGN). In contrast, Tac-furin passes through maturing endosomes on its way to the TGN without accumulating to any observable degree in the endocytic recycling compartment. Dashed line indicates anterograde transport from TGN to PM. Above each panel are diagrams of the Tac chimeras showing the Tac lumenal (white) and transmembrane (black) domains, and the cytoplasmic tail domain of furin or TGN38 (gray), respectively. <b>C</b>) Stick representation of LMB-2 indicating the various domains of the immunotoxin. The variable domain (heavy and light chain) of the Tac antibody (gray) are linked with a small peptide linker. The truncated version of <i>Pseudomonas</i> exotoxin A (white) consists of the translocation domain (II) and the enzymatic domain (III), but lacks the wild type binding domain. The gray arrow indicates an essential proteolytic processing site after amino acid 279.</p

Effects of heterogeneous Tac expression on LMB-2 cytotoxicity.

<p>Tac-TGN38 (<b>A</b>) and Tac-furin (<b>B</b>) cells were treated for 24 hours with doses of LMB-2 up to 10 ng/ml. Cells were then fixed, permeabilized and stained with anti-Tac monoclonal antibody and Alexa546 conjugated secondary antibody. The histograms show the frequency distribution of Tac intensity for each cell line after exposure to varying concentrations of LMB-2. The experiments were replicated at least 3 times for each cell line, and representative data from one experiment are shown. <b>C</b>) Tac-TGN38 and Tac-furin cells were fixed and then stained with Hoechst dye, anti-Tac monoclonal antibody and subsequently with Alexa546 conjugated secondary antibody. The average fluorescence per cell was measured as described in Methods. Images were subjected to background and shade correction before intensity calculations. Fluorescence intensity per field was divided by the cell number to calculate the average intensity per cell. The data are the average of 9 experiments. The standard error of the mean is shown.</p