Autophagy and self-renewal after plumbagin treatment.

<p><b>(A)</b> Time dependent dynamics of CYTO-ID Green (CYTO-ID+, CYTO-ID++) after 2 μM plumbagin treatment. See autophagy peak at 8h-time point. <b>(B)</b> Time dependent dynamics of cell deaths after 2 μM plumbagin treatment; total CytoID staining (CYTO-ID+ and CYTO-ID++) depicts autophagy, AnnexinV+/PI- depicts apoptosis or early oncosis, and AnnexinV+/PI+ depicts necrosis (raw data and gating strategy in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145016#pone.0145016.s002" target="_blank">S2 Appendix</a>). <b>(C)</b> Amount of autophagic cells after bafilomycin treatment; red cluster depicts CYTO-ID++ population, blue cluster Cyto-ID+ population. <b>(D)</b> Amount of autophagic cells in control (not-treated population); red cluster depicts CYTO-ID++ population, blue cluster CYTO-ID+ population. <b>(E)</b> Amount of autophagic cells after plumbagin treatment; red cluster depicts CYTO-ID++ population, blue cluster CYTO-ID+ population. <b>(F)</b> Western blot for LC3-I and LC3-II isoforms at 12 time-points after 2 μM plumbagin treatment (raw data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145016#pone.0145016.s003" target="_blank">S3 Appendix</a>). <b>(G)</b> Graphic representation of western blot results for LC3-I and LC3-II isoforms at 12 time-points after 2 μM plumbagin treatment. <b>(H)</b> Time dependent dynamics of <i>POU5F</i>, <i>SOX2</i>, <i>NANOG</i>, and <i>BECN1</i> gene expression after 2 μM plumbagin treatment. <b>(I)</b> Time dependent dynamics of <i>BIRC5</i>, <i>HIF1A</i>, <i>CCL2</i>, and <i>MAP1LC3</i> gene expression after 2 μM plumbagin treatments. <b>(J)</b> Principal component analysis—projection of variables on the two-factor plane. See distinct clustering of genes with flow-cytometric measurements based on metabolic stress and reprogramming (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145016#sec018" target="_blank">results</a>). <b>(K)</b> Principal component analysis—projection of time-points on the two-factor plane. The first and the second factor are designated as “metabolic stress high-low” and “non-reprogrammed-reprogrammed”, respectively (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145016#sec018" target="_blank">results</a>). <b>(L)</b> Cluster analysis of gene expression. The “reprogramming cluster”involved <i>POU5F</i>, <i>SOX2</i>, <i>NANOG</i>, and <i>CCL2</i>; the “autophagic and hypoxia cluster”involved <i>HIF1A</i>, <i>MAP1LC3</i>, and <i>BECN1</i>; the third cluster involved <i>BIRC5</i> gene. <b>(M)</b> Cluster analysis of gene expression of 12 time-points after 2 μM plumbagin treatment and PGCCs selected by hypoxia and starvation; based on correlations of gene expression patterns, similarity between PGCCs and PC-3 cells after 20h plumbagin treatment was found. <b>(N)</b> Morphology of cells after 20h plumbagin treatment; cells with polyploid giant cancer cell (PGCCs)-like morphology are highlighted by arrows. <b>(O)</b> Morphology of PC-3 cells after 1 month of hypoxia and starvation; cells with PGCCs-like morphology are highlighted by arrows.</p

Effect of plumbagin treatment on viability and size of cells.

<p><b>(A)</b> Real-time monitoring of relative cell impedance (showed as a cell index) using the RTCA system. <b>(B)</b> MTT-assessed response to 6h plumbagin treatment. <b>(C)</b> MTT-assessed response to 24h plumbagin treatment. <b>(D)</b> IC50 values according to RTCA and MTT and length of treatment. <b>(E)</b> Time-dependent changes in the quantity of large cells with intact nuclei (SYTO 16++/FSC+) and intact cells (AnnexinV-/PI-) assessed by flow-cytometry. <b>(F)</b> Numbers of large healthy cells depicted as SYTO 16++ /FSC+ (red) cluster at flow-cytometric dot plot at 6h time-point; Forward-scattered light (FSC) is proportional to cell-surface area or size. <b>(G)</b> Granularity of large healthy cells depicted as SYTO 16++ /SSC+ (red) cluster at flow-cytometric dot plot at 6h time-point; Side-scattered light (SSC) is proportional to cell granularity or internal complexity. <b>(H)</b> Morphology of PC-3 cells after 6h plumbagin treatment, 20x magnification, phase contrast microscopy. <b>(I)</b> Numbers of large healthy cells depicted as SYTO 16++/FSC+ (red) cluster at flow-cytometric dot plot at 24h time-point; Forward-scattered light (FSC) is proportional to cell-surface area or size. <b>(J)</b> Granularity of large healthy cells depicted as SYTO 16++/SSC+ (red) cluster at flow-cytometric dot plot at 24h time-point; Side-scattered light (SSC) is proportional to cell granularity or internal complexity. <b>(K)</b> Morphology of PC-3 cells after 24h plumbagin treatment; giant PC-3 cells with polyploid giant cancer cell (PGCCs)-like morphology are highlighted by arrows. 20x magnification, phase contrast microscopy.</p

Time-lapse of cell interactions.

<p>For detailed time-lapse Videos see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145016#pone.0145016.s004" target="_blank">S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145016#pone.0145016.s007" target="_blank">S4</a> Videos. <b>(A)</b> Time-lapse imaging of entosis; internalized cell (red arrow) played an active role in its engulfment, which resulted in complete internalization. Both types of cells (engulfing and engulfed) were viable for a long time and lived by about five hours longer than the other observed plumbagin-treated tumour cells. <b>(B)</b> Time-lapse imaging of cell fusion with cannibalism (digestion of engulfed cell); during fusion-cannibalism of living cells, the cannibalic cell (red arrow) came in contact with the target cell (blue arrow). The next step was gradual engulfment of the target cell. The nucleus of the target cell appeared initially unaltered whereas the engulfing cell’s nucleus began to change into a semilunar shape. Bird eye structure was observed as a consequence of target cell vacualisation (see green arrow). <b>(C)</b> Time-lapse imaging of cannibalism without fusion; the dying cell (blue arrow) was attacked and exploited by the cannibalic cell (red arrow). The target cell was dead after the attack. <b>(D)</b> Time-lapse imaging of oncosis; oncotic cells formed typical cytoplasmic blebs that usually lead to necrosis (see red arrow). <b>(E)</b> Time-lapse imaging of reverse oncosis; initial forming of oncotic blebs (see red arrow) did not lead to necrosis; the bleb was absorbed and the cell remained viable. (<b>A-E</b>) Multimodal holographic microscopy, 10x magnification. <b>(F)</b> Communication between PC-3 cells; visualised by TEM (see red arrows). <b>(G)</b> Vesicular transfer between PC-3 cells; visualised by TEM (see red arrows). <b>(H)</b> Speed of the migration of untreated PC-3 cell population; assessed from holographic microscopy data by CellProfiller software by measurement of “distance travelled”parameter. <b>(I)</b> Speed of the migration of PC-3 cell population after 2 μM plumbagin treatment.</p

Possible cell fate under oxidative stress.

<p>Prolonged oxidative stress (ROS) leads to severe cell damage and depletion of cell energy reserves. Nevertheless, many cell injuries caused by ROS could be sublethal, especially because PC-3 cells have non-functional p53 and therefore disrupt the triggering of apoptosis. Even if a damaged cell is driven to oncosis, reversion of this process is possible, particularly if the cell is able to restore ATP production. A way to gain enough energy for survival could be autophagy. Similar to autophagy, digestion of the cytoplasm of neighbouring cells can provide a source of amino acids. Retention of a foreign nucleic acid by cannibalistic engulfment could result in aneuploid or polyploid state. Furthermore, reduction in membrane and cell stiffness due to protein catabolism by autophagy could reflect increased entotic activity. Cell in cell structure results in the decreased surface-to-volume ratio, thereby minimizing cell membrane requirements. Furthermore, a live cell internalized by entosis could disrupt host cell division. Subsequently, cytokinesis often fails, which can lead to the formation of polyploid giant cancer cells (PGCCs). PGCCs often dye by apoptosis or senescence, but a small fraction of these cells is able to survive and even produce aneuploid progeny. Senescence, polyploidy and self-renewal seem to be three steps to immortality of cancer cells. Autophagy could play an important role in all of them.</p

Mechanistic characterization of engulfed and engulfing cells in entosis.

<p><b>(A)</b> Trajectory travelled of both engulfing and engulfed cell until cell fusion. See differences in the travelled distance and in directionality of individual cells. Directionality describes "purposefulness" of the movement where 0% indicate random movement and 100% indicate straight line trajectory between starting and ending position. Position (0.0, 0.0) indicate place of cell fusion. <b>(B)</b> Changes in cell mass and <b>(C)</b> cell area of engulfed and engulfing cell.</p

Reactive oxygen species (ROS)-induced mitophagy.

<p><b>(A)</b> Phase contrast microscopy of PC-3 cell after plumbagin treatment. <b>(B)</b> General accumulation of ROS after plumbagin treatment monitored by confocal microscopy by using CellROX Deep Red Reagent. Areas with ROS accumulation are highlighted by arrows. <b>(C)</b> Mitochondria staining monitored by confocal microscopy using MitoTracker Green; area associated with ROS in Fig 2B are highlighted by arrows. <b>(D)</b> Endoplasmic reticulum (ER) staining monitored by confocal microscopy using ERTracker Red; areas associated with ROS in Fig 2B are highlighted by arrows. <b>(E)</b> Untreated PC-3 cell, cross-section of undamaged mitochondria (highlighted by red arrow); Transmission Electron Microscope (TEM) visualization. <b>(F)</b> plumbagin-treated PC-3 cell, mitochondria coated by ER membrane with ribosomes (highlighted by red arrow); TEM visualization. <b>(G)</b> Plumbagin-treated PC-3 cell, gradual degradation of mitochondria in autophagosomes visualised by TEM (red arrows); Swollen mitochondria as a marker of damage (yellow arrow).</p

Holographic mode setup in Multimodal holographic microscope is based on the Mach-Zehnder-type interferometer.

<p>The light is divided into two separate optical paths—object arm and interferometer reference arm. Both arms consist of condenser (C), objective (O) and tube lens (TL). In the reference arm, a diffraction grating (DG) is placed. The object beam and the reference beam recombine in the output plane and create interference fringes pattern, which is captured by the camera (D). S—source; CL—collector lens; BS—beam splitter; M—mirror; C—condenser; O-objective; TL—tube lens; DG—diffraction grating; OL—output lens; D—detector.</p

Estimation of oncosis progression by multimodal holographic microscopy.

<p>Annexin V (green) and propidium iodide (PI, red) staining. Initial step of oncosis (first row, red arrow) is annexin V−/PI− and thus distinguishable only by native morphology, see typical cytoplasmic bleb in the phase image. This causes false-negativity by flow-cytometry. Second, early oncotic cells feature larger blebs and are annexin V+/PI−. Late oncosis is double positive for staining, with no apparent karyolysis. Advanced oncosis/necrosis transition is typical by double-positive staining and karyolysis.</p

Morphology of Apoptotic, necrotic and oncotic cells.

<p><b>A.</b> Characteristic apoptotic, necrotic and oncotic cells in multimodal holographic microscope, simulated DIC (differential interference contrast). 20 × magnification was used in MHM. Annexin V staining for the verification of cell membrane alteration. Red arrow indicates annexin V-positive “advanced” oncotic cell. Apoptotic cells displayed in initial step (left) with the typically round-shaped cells and in advanced step with the formation of apoptotic bodies. <b>B.</b> Scheme of typical apoptotic, necrotic and oncotic cells. Typical characteristics visible by MHM phase image. For a detailed description of the characteristic features of apoptotic, necrotic, and oncotic cells, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121674#pone.0121674.t001" target="_blank">Table 1</a>.</p