Effects of dynamin II knockdown on pDNA electrotransfection.

<p>B16-F10 cells were transfected with either the control siRNA or one of the two specific siRNA oligos directed against two different sequences (i.e., Sq1 and Sq2) in mouse dynamin II gene for silencing its expression. The siRNA treatment was followed by a 48 hr incubation period prior to pDNA electrotransfection. Dynamin II and β-actin (loading control) expression levels in Western blot analysis are shown in Panel A and normalized electrotransfection efficiencies are shown in Panel B. The bars and error bars indicate the means and standard deviations of 4 independent trials, respectively. The data from each trial, used in mean and standard deviation calculation, was the average value of replicates or triplicates. *, P<0.05 (Mann-Whitney U test).</p

Dependence of membrane-bound pDNA on cation concentrations.

<p>pDNA was labeled with YOYO-1 dye with basepair-to-dye ratio of 5∶1. The binding was characterized in terms of (A) percent of pDNA-associated cells and (B) average fluorescence intensity with arbitrary unit (a.u.) per pDNA-associated cell. The number of independent trials (n) was 5–6. The symbols and error bars denote means and standard deviations, respectively.</p

Dependence of electrotransfection efficiency on cation concentrations.

<p>eTE is defined as the percent of live cells expressing GFP. B16-F10 cells were electrotransfected (400 V/cm, 5 msec, 8 pulses, 1 Hz) with unlabeled GFP-encoding pDNA in a transfection buffer. GFP expression was measured using flow cytometry after 24 hr incubation. (A) The low ionic strength medium supplemented with Ca²⁺ or Mg²⁺ at varying concentrations was used as the electrotransfection buffer. n = 7–8. The symbols and error bars denote means and standard deviations, respectively. The peak eTE value in each curve was significantly higher than those at both ends of the same curve (P<0.05). In Panels (B) and (C), OptiMEM was used as the electrotransfection buffer. After 20 min incubation post electrotransfection, the cells were re-suspended in the low ionic strength medium supplemented with either Ca²⁺ or Mg²⁺ at varying concentrations and treated again with the same electric field. The GFP expression was quantified at 24 hr. n = 4. The filled circles denote data from individual samples, the “x” symbol represents the mean of the samples at a given cation concentration, and the line represents the linear regression of the mean data. The mean value was statistically independent of the variation in Ca²⁺ and Mg²⁺ concentrations (P>0.05, Mann Whitney U test).</p

Reduction in cellular uptake of pDNA and the eTE by endocytic inhibitor treatment.

<p>pDNA covalently labeled with rhodamine (red) was electrotransfected (400 V/cm, 5 msec, 8 pulses, 1 Hz) into cells pre-treated with (A) DMSO (drug vehicle) or (B) dynasore (80 µM) for 1 hr. After electrotransfection, the cells were incubated at 37°C to enable cellular uptake of pDNA for 30 min. At the end of incubation, the cells were examined using confocal microscopy. Arrows in the microscopic images denote pDNA internalized by cells. To visualize three-dimensional distribution of pDNA in the cytosol, two optical cross-sections of DMSO-treated cells in x–z and y–z planes are shown in Panel (C). Effects of endocytic inhibitor treatment on the eTE are shown in Panel (D). Cells were treated with DMSO (Ctrl), 28 µM CPZ, 200 µM genistein (GE), or 80 µM dynasore (DN) for 1 hr prior to electrotransfection with the GFP-encoding pDNA. The eTE, defined as the percent of live cells expressing GFP, was quantified after cells were cultured at 37°C for 24 hr. n = 4–6. * P<0.05 and ** P<0.005 (Mann-Whitney U test).</p

Effects of trypsin treatment on pDNA adsorption to cell membrane and eTE.

<p>(A) YOYO 1-labeled pDNA (green) formed complexes with FM4-64FX labeled plasma membrane (red) following exposure of cells to pulsed electric field (400 V/cm, 5 msec, 8 pulses, 1 Hz). The image was taken shortly after the application of electric field. (B) The experimental protocol was the same as that in the Panel (A), except that at 10 min post electric field exposure, the cells were treated with 0.25% trypsin-EDTA solution for 30 min at 37°C. The image was taken after the trypsin treatment. (C) B16-F10 cells in pDNA solution were exposed to the same electric pulses (EP) as above. At 10 or 40 min post EP exposure, the cells were treated with 0.25% trypsin-EDTA for 30 min at 37°C. Then, the cells were cultured for 24 hr at 37°C. The eTE was measured as the percent of live cells expressing GFP and normalized by the data from the untreated group. The solid column and error bar represent mean and standard deviation of the relative eTE, respectively. n = 6–9. * P<0.05 (Mann-Whitney U test).</p

Mathematical model of the Phoenix Rising pathway.

<p>Seventeen molecular interactions in apoptotic cells were considered in the model. The input of the model was radiation-induced activation of C3, C7, and NFκB. Through the pathway, the activated molecules could result in secretion of PGE2 into the cell's microenvironment. The secreted PGE2 may promote proliferation of stem cells, progenitor cells, and tumor cells in surrounding regions. The mathematical model was used to simulate the dynamics of PGE2 synthesis and how inhibitions of different molecular players in the pathway affected PGE2 synthesis.</p

Model simulation of time-dependent changes in [iPLA2], [iPLA2^*], [AA], and [PGH2] after 10-Gy radiation.

<p>The simulation was performed for C3 knockout MEF cells.</p

Dependence of simulated [PGE2] on k₁ and k₃ in MEF cells.

<p>The value of k_i (i = 1, 3) was normalized to its baseline shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003461#pcbi-1003461-t003" target="_blank"><b>Table 3</b></a>. [PGE2] was calculated at 48 hours after radiation. Four scenarios were simulated in the study: (i) dependence of [PGE2] on k₁ when k₃ was set at its baseline, (ii) dependence of [PGE2] on k₃ when k₁ was set at its baseline, (iii) dependence of [PGE2] on k₁ in C7 KO cells, and (iv) dependence of [PGE2] on k₃ in C3 knockout (KO) cells.</p

Effects of COX2 inhibition on model simulated [PGE2] in wild type MEF cells at 48 hours post radiation.

<p>Experimentally, COX2 inhibition can be achieved through treatment of cells with exogenous competitive inhibitors. In this study, the competitive inhibition was modeled through introducing a parameter, α, which was the ratio of inhibitor concentration versus equilibrium constant of binding between COX2 and its inhibitor. It can be observed that [PGE2] decreased rapidly with increasing the value of α.</p

Model simulation of time-dependent changes in [iPLA2], [iPLA2^*], [AA], and [PGH2] after 10-Gy radiation.

<p>The simulation was performed for wild type 4T1 cells.</p