Effects of continuous X-ray irradiation with different peak energies and dose rates on the proliferation of <i>C. neoformans</i>.

<p>As discussed in the main text, radiation effects were quantified by calculating the radiation-induced change (Q_e) in CFU/ml, normalized by the initial cell concentration, as follows: Q_e = (X_r(i) – X_c)/X₀, where X_r(i) is the CFU/ml for each irradiated culture, X_c is the mean CFU/ml for corresponding unirradiated controls, and X₀ is the mean CFU/ml at the start of irradiation. Q_e represents the number of extra descendants produced by each founder cell due to the effects of irradiation on cell proliferation.</p

Analysis of the proliferation of <i>C. neoformans</i> continuously X-irradiated for 24 hours.

<p>The symbols represent radiation-induced change (Q_e) in CFU/ml (normalized by the initial cell concentration) measured for melanized and non-melanized cells at each combination of X-ray peak energy (150 or 320 kVp) and dose rate. Different symbol colors, shapes, and bar colors represent different experiments. Horizontal bars indicate mean values from each experiment. The lines are best-fit predictions generated by the proposed model, Q = A(e, t, m) – [B(e, t, m) x R], which predicts the radiation effect on proliferation relative to background conditions (Q, shown on the y-axis), based on radiation dose rate (R shown on the x-axis), X-ray peak energy (e), duration of irradiation (t), and cell melanization status (m). Model parameter A represents radiation-induced growth enhancement at low dose rates, and parameter B represents radiation-induced growth inhibition, proportional to dose rate.</p

Effects of 24 and 48 hours of continuous X-ray irradiation on the proliferation of <i>C. neoformans</i>.

<p>The symbols represent mean values of the radiation-induced change (Q_e) in CFU/ml (normalized by the initial cell concentration) for the same X-ray dose rate categories as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085561#pone-0085561-t001" target="_blank">Table 1</a>, plotted as function of mean dose rate for each category. Error bars represent standard errors. The abbreviations “mel +” and “mel –” represent melanized and non-melanized cells, respectively.</p

Analysis of the proliferation of <i>C. neoformans</i> continuously X-irradiated for 48 hours.

<p>Interpretations for the axes, symbols, and curves are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085561#pone-0085561-g003" target="_blank">Figure 3</a>.</p

Analysis of best-fit model parameter values.

<p>The proposed model, Q = A(e, t, m) – [B(e, t, m) x R], which predicts the radiation effect on proliferation relative to background conditions (Q), based on radiation dose rate (R), X-ray peak energy (e), duration of irradiation (t), and cell melanization status (m), was fitted to 10,000 Monte Carlo simulated data sets, generated from each observed data set by nonparametric bootstrapping. This procedure was repeated for 8 observed data sets, which represent combinations of 150 or 320 kVp X-ray peak energy, 24 or 48 hour exposure duration, and positive or negative cell melanization status (mel+or mel –). Each “cloud” of points represents the spread of values for model parameters A and B which are consistent with the corresponding data set, randomly varied by bootstrapping. Black open symbols superimposed on each “cloud” represent best-fit parameter values to the observed data (unperturbed by bootstrapping).</p

Radioimmunotherapy of Hep 3B2.1-7 tumors in nude mice:

<p>a) changes in tumor volume; b) control untreated mouse; c) mouse treated with 600 µCi ¹⁸⁸Re-4H9 mAb. Both mice shown on Day 18 post-treatment. In b and c lower panels show H&E stained tumors at the completion of the experiment.</p

Detection of viral antigens in tumor cells and in tumors:

<p>a, b) immunofluorescence of fixed and permeabilized tumor cells. Left panels show light microscopy images of the cells. Heavily damaged cells are marked with arrows. Right panels show immunofluorescent images of the same slides treated with viral protein-specific mAbs followed by FTIC-conjugated polyclonal antibody to mouse IgGs: a–CasKi cells and E6-specific C1P5 mAb, b-Hep 3B2.1-7 cells and HBx-specific 4H9 mAb; c) immunohistochemistry of CasKi tumors. Left panel shows binding of E6-specific mAb C1P5. Right panel shows absence of binding of control mAb 18B7; d) western blot of Hep 3B2.1-7 tumor with HBx-specific mAb 4H9.</p

Scintigraphic images of tumor-bearing mice 24 hours post-injection with: a) CasKi tumor, E6-specific mAb ¹⁸⁸Re-C1P5 mAb; b) CasKi tumor, control ¹⁸⁸Re-18B7 mAb; c) Hep 3B2.1-7 and A2058 human metastatic melanoma tumors, HBx-specific ¹⁸⁸Re-4H9 mAb.

<p>Scintigraphic images of tumor-bearing mice 24 hours post-injection with: a) CasKi tumor, E6-specific mAb ¹⁸⁸Re-C1P5 mAb; b) CasKi tumor, control ¹⁸⁸Re-18B7 mAb; c) Hep 3B2.1-7 and A2058 human metastatic melanoma tumors, HBx-specific ¹⁸⁸Re-4H9 mAb.</p

Expression of E6 and E7 in human cervical carcinoma cell lines and of HBx in hepatocellular carcinoma cell line by Western blot and effect of MG132 proteasome inhibitor treatment on the levels of E6, E7 and HBx in these cell lines:

<p>a) E6 from protein extracts of CasKi cells treated with MG132 for 3 hrs; b) E7 from protein extracts of CasKi cells treated with MG132 for 3 hrs; c) E6 from protein extracts of CasKi cells treated with MG132 for 6 hrs; d) E7 from protein extracts of SiHa cells treated with MG132 for 3 hrs; e) E7 from protein extracts of HeLa S3 cells treated with MG132 for 3 hrs; f) HBX from protein extracts of Hep 3B2.1-7 cells treated with MG132 for 3 hrs.</p

ESR spectra of melanin derived from <i>C. sphaerospermum</i> grown on different substrates: a) potato dextrose agar; b) Sabaroud dextrose agar; c) potato dextrose agar impregnated with 25 µg/mL tricyclazole.

<p>Differences in <i>C. sphaerospermum</i> ESR spectra in comparison with <i>C. neoformans</i> are marked with arrows. ESR spectra were obtained by suspending “ghosts” in water. Ordinate is the derivative of the ESR absorption in arbitrary units. Cs - <i>C. sphaerospermum.</i></p