Comparison of motile activity of BC and LC cells.

<p>(<b>A</b>) Composite trajectories of BC and LC cells migrating under isotropic conditions are shown as circular diagrams. In each diagram, the initial point of each trajectory was placed at the center of the circle. Each trajectory was constructed from 120 (BC) or 48 (LC) successive positions of cell centroids recorded at 15-sec (BC) or 150-sec (LC) time intervals, respectively. The movement of BC and LC was recorded for 30 minutes and 150 minutes, respectively. (<b>B</b>) Diagram showing the speed of cell movement and speed of cell displacement (n = 50); *Statistically significant (p<0.05).</p

The role of intracellular Ca²⁺ and myosin II in electrotaxis of BC and LC cells.

<p><b>(A)</b> Composite trajectories of BC and LC cells migrating in the presence of dcEF (3 V/cm) and 30 μM BAPTA-AM (chelator of [Ca^<b>2+</b>]_i), 10 μM ML-7 (MLCK inhibitor) or 50 μM blebbistatin (myosin II inhibitor) shown as circular diagrams. In each diagram, the initial point of each trajectory was placed at the center of the circle. The x-axis corresponds to the direction of the electric field. The cathode was always placed at the right side of the diagram. Each trajectory was constructed from 120 (BC) or 48 (LC) successive positions of cell centroids recorded at 15-sec (BC) or 150-sec (LC) time intervals, immediately after exposure of cells to dcEF. The movement of BC and LC cells was recorded for 30 minutes and 150 minutes, respectively (n = 50). (<b>B</b>) The diagrams depict the values of directional cosines γ. *Statistically significant vs. 3 V/cm (p<0.05).</p

The effect of Rac, Cdc42, Rho and ROCK inhibitors on electrotaxis of BC and LC cells and the effect of dcEF on the activity of small Rho GTP-ases.

<p><b>(A)</b> Composite trajectories of BC and LC cells migrating in the presence of dcEF (3 V/cm) and 50 μM NSC23766 (Rac1 inhibitor), 50 μM ZCL278 (Cdc42 inhibitor III), 30 μM Rhosin (Rho inhibitor) or 10 μM Y-27632 (ROCK inhibitor) are shown as circular diagrams. In each diagram, the initial point of each trajectory was placed at the center of the circle. The x-axis corresponds to the direction of the electric field. The cathode was always placed at the right side of the diagram. Each trajectory was constructed from 120 (BC) or 48 (LC) successive positions of cell centroids recorded at 15-sec (BC) or 150-sec (LC) time intervals, immediately after exposure of cells to dcEF. The movement of BC and LC was recorded for 30 minutes and 150 minutes, respectively (n = 50). (<b>B</b>) The diagrams depict the values of directional cosines γ. (<b>C</b>) The activity of Rac1, Cdc42 and RhoA in BC and LC cells under isotropic condition and after exposition of cells to dcEF for 5 and 15 minutes determined by the G-Lisa assay. *Statistically significant vs. 3 V/cm (p<0.05).</p

The morphology of two sublines of Walker carcinosarcoma WC 256 cells migrating in an adhesive mode but forming different protrusions.

<p><b>(A, C)</b> Weakly adherent cells forming blebs spontaneously (BC); <b>(B, D)</b> Strongly adherent cells forming lamellipodia (LC); photographs obtained in DIC optics <b>(A, B)</b> or in SEM <b>(C, D).</b> Arrows indicate blebs at the leading edges of cells.</p

A diagram showing all proteins regulated in proton-irradiated BLM cells.

<p>The level of 17 proteins changed (>1.5×) in comparison with control. Thirteen proteins were upregulated (in black) and 4 were downregulated (in red). Here they are presented in four, not exclusive, groups: i) DNA repair and stress, ii) proliferation and survival control, iii) metabolic and iv) connected to motility and the cytoskeleton. ACTN 4 - α Actinin 4, Caprin-1 - Cytoplasmic activation/proliferation-associated protein-1, FAB-2 - Far upstream element binding protein 2, G3BP1 - RasGAP SH3-domain-binding protein 1, GADPH - Glyceraldehyde 3-phosphate dehydrogenase, MCM-7– Minichromosome Maintenance Protein 7, Moesin - Actin-regulatory protein, MVP - Major Vault Protein, PDCD6 - Programmed cell death 6, or apoptosis-linked gene-2, STRAP - Serine-threonine kinase receptor-associated protein, TIM - Triosephosphate isomerase, VCP – Transitional endoplasmic reticulum ATPase.</p

The zoomed changes in the level of spots identified by MS in the two groups.

<p>Left panel – control group, Right panel –3 Gy irradiated group. Spot numbers as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084621#pone-0084621-t001" target="_blank">Table 1</a>.</p

The effect of dcEF on the migration of BC and LC cells.

<p><b>(A)</b> Composite trajectories of BC and LC cells migrating in the absence and in the presence of dcEFs are shown as circular diagrams. In each diagram, the initial point of each trajectory was placed at the center of the circle. The x-axis corresponds to the direction of the electric field. The cathode was always placed at the right side of the diagram. Each trajectory was constructed from 120 (BC) or 48 (LC) successive positions of cell centroids recorded at 15-sec (BC) or 150-sec (LC) time intervals, immediately after the exposure of cells to dcEF. The movement of BC and LC was recorded for 30 minutes and 150 minutes, respectively. (<b>B</b>) Diagrams presenting the values of directional cosines γ for BC and LC cells depending on applied field strength; mean ± SEM; p<0.05 vs 0 V/cm (<b>C</b>) The dynamics of reversibility of the direction of cell movement. The directionality of cell movement was completely reversible upon reversing the field polarity (3V/cm). For both cell sublines the average directional cosines γ were analyzed every 5 minutes. Perpendicular lines indicate the time when the electric field polarity was reversed (n = 50). *Statistically significant vs. 0 V/cm (p<0.05).</p

The effect of proton beam irradiation on BLM melanoma cells.

<p>A. Proliferation of BLM cells after proton beam irradiation with doses 0 (open circle), 2 (cross), 3 (diamond) and 4 Gy (triangle). Cells were irradiated in suspension, and then plated in 96-well plates. B, C. DNA damage in untreated (white bar), and irradiated with 3 Gy of proton beam (stripped bar) BLM cells are presented in terms of the percentage of DNA that left the comet's head and was found in the comet’s tail after electrophoresis (TDC).</p

Distribution of F-actin in migrating BC and LC cells.

<p>Cells were transfected with LifeAct, a 17-amino-acid peptide, which stains F-actin in eukaryotic cells. (<b>A</b>) Sequence of frames showing bleb formation (indicated by arrow) in weakly adherent BC cells. <b>(B)</b> Strongly adherent lamellipodia forming LC cell.</p

Signaling pathways involving proteins upregulated after sublethal proton beam irradiation.

<p>Signaling pathways involving proteins upregulated after sublethal proton beam irradiation.</p