HUVEC confocal immunofluorescent microscopy images LC3A(green)/LC3B(red) (Fig. 1a), LC3A(green)/LAMP2a(red) (Fig. 1b), LC3B(red)/CathepsinD(green) (Fig. 1c) and p62(red) (Fig. 1d).

<p>Co-localization is presented as yellow spots. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g001" target="_blank">Figure 1a</a>: autophagosomes stained for LC3A (perinuclear localization) and LC3B (cytoplasmic localization). Irradiation-induced accumulation of both LC3A and LC3B autophagosomes at 4 and 8 post-irradiation days (1a2,1a3). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g001" target="_blank">Figures 1b</a>1 and 1c1 show an evident degree of co-localization of LC3A with LAMP2a and of LC3B with CathepsinD. At 4 and 8 post-irradiation days there was a lack of co-localization, which suggests autophagy flux suppression (1b2, 1b3, 1c2, 1c3). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g001" target="_blank">Figure 1d</a> shows poor cytoplasmic and intense nuclear presence of p62 in control cells (1d1); intense accumulation of p62 in the cytoplasm was evident 4 and 8 days after irradiation (1d2,3). Graphic presentation of fluorescent intensity of the above immunostaining is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g001" target="_blank">Figure 1e</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g001" target="_blank">Figure 1f</a> shows the western blot analysis in the pellet fraction of HUVEC.</p

Confocal images of HUVEC cultured cells after exposure to 2 Gy of IR, with and without 2 day pre-incubation with SMER28 (Fig. 4a).

<p>LC3A(green)/LC3B(red) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g003" target="_blank">Fig. 3a1,2</a>), LC3A(green)/LAMP2a(red) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g003" target="_blank">Fig. 3a3,4</a>), LC3B(red)/CathepsinD(green) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g003" target="_blank">Fig. 3a5,6</a>) and p62(red) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g003" target="_blank">Fig. 3a7,8</a>). Co-localization is shown as yellow structures. Western blot images from supernatant and pellet protein fractions is shown in Fig. 4b.</p

HUVEC confocal images LC3A(green)/LC3B(red) (Fig. 3a1,2), LC3A(green)/LAMP2a(red) (Fig. 3a3,4), LC3B(red)/CathepsinD(green) (Fig. 3a5,6) and p62 (red) (Fig. 3a7,8), before and after 2 days exposure to SMER28.

<p>Co-localization is shown as yellow structures. SMER28 induces expression of LC3A/LC3B and co-localization with lysosomal markers, while p62 cytoplasmic content is reduced. Graphic presentation of fluorescent intensity of the above immunostaining is shown in Fig. 3b. Figure 3c shows western blot images from the supernatant or pellet of HUVEC proteins.</p

HUVEC confocal images of Beclin 1 (green) (Fig. 2a), ATF4 (green) (Fig. 2b) and HIF2α (red) (Fig. 2c).

<p>Intense accumulation of Beclin 1 was evident 4 and 8 days after irradiation (2a2, 2a3). Intense nuclear localization of ATF4 (arrows) at 4 and 8 post-irradiation days is shown in figure panel 2b, while HIF2α was also induced in the cytoplasm and nuclei (arrows) of HUVEC (figure panel 2c). Graphic presentation of fluorescent intensity of the above immunostaining is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g001" target="_blank">Figure 1d</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102408#pone-0102408-g001" target="_blank">Figure 1e</a> shows the western blot analysis in the pellet fraction of HUVEC.</p

Confocal microscopy after γΗ2ΑX immunostaining, showing red foci in the nuclei of control (6a1,6a3) and irradiated cells (6a2,6a4), with (6a3,6a4) and without (6a1,6a2) pre-incubation with SMER28 (Fig. 6a).

<p>Computerized calculation of γH2AX foci in the nuclei of cells. Statistical significance refers to comparison with control cells (Fig. 6b).</p

Confocal microscopy for LC3A/LC3B, specific suppression of LC3A (Fig. 5a2) and of LC3B (Fig. 5a3) after incubation with relevant LC3 siRNAs (Fig. 5a).

<p>Radiation dose and response curves confirm a shift to the right when a suppression of the LC3A/LC3B expression is achieved before irradiation, while the curve shifts to the right when cells are pre-incubated with non-toxic doses of the mTO- independent autophagy inducer SMER28 (Fig. 5b). Viability curves of HUVEC cells after exposure from 1–3 days at various SMER28 concentrations confirm nontoxic effect at 25 µM (Fig. 5c). Radiation dose and recovery curves comparing the cell population at the nadir (8^th day) vs. exponential growth onset (12^th day) show enhanced recovery potential when cells are pre-incubated with SMER28 (Fig. 5d).</p

Mast cells co-expressing CD68 and inorganic polyphosphate are linked with colorectal cancer

<div><p>Inflammation is a hallmark of colorectal cancer (CRC). Neutrophils are well-known mediators in tumor biology but their role in solid tumors, including CRC, was redefined by neutrophil extracellular traps (NETs). Given that it was recently demonstrated that platelet-derived polyP primes neutrophils to release NETs, we examined surgical specimens from CRC to investigate the presence of polyP, as a possible NET inducer. Biopsies with adenomas, hyperplastic polyps, inflammatory bowel disease and healthy colon tissues were used as controls. In all cases, the presence of polyP was apparent, with the main source of polyP being the mast cells. In all CRC and all adenomas with high-grade dysplasia, a substantial number of mast cells, more than 50%, co-expressed intracellularly polyP with CD68 surface antigen (CD68+), but this was not the case in the other examined disorders. PolyP-expressing mast cells were detected in close proximity with tumor cells and neutrophils, suggesting polyP expression by CD68+ mast cells among the stimuli which prime neutrophils to release NETs, in CRC. Moreover, the detection of CD68+ polyP-expressing mast cells could represent a potential prognostic marker in colorectal adenomas and/or carcinomas.</p></div

CD68+ mast cells are important sources of polyP in CRC and adenomas with high-grade dysplasia, and are localized next to neutrophils in CRC.

<p>Confocal microscopy for <b>(A)</b> Tryptase / CD68, <b>(B)</b> polyP / NE and <b>(C and D)</b> polyP / CD68 staining in sections of <b>(A and B)</b> colonic adenocarcinoma specimens and adenomas with <b>(C)</b> high- or <b>(D)</b> low-grade dysplasia. Blue: PI, Green: (A) mast cell tryptase or (B and D) JC-D8 polyP-specific fluorescent probe, Red: (A, C and D) CD68 or (B) NE. One representative out of (A and B) ten or (C and D) six independent experiments is shown. Original magnification x600. Scale bar– 5μm. HG–high grade, LG–low grade.</p

Identification of the polyP-expressing cells in CRC, in pre-malignant and in non-malignant conditions of the colon.

<p>Identification of the polyP-expressing cells in CRC, in pre-malignant and in non-malignant conditions of the colon.</p

PolyP-expressing cells are positive for mast cell tryptase and CD68 in human colon cancer.

<p>Confocal microscopy for <b>(A)</b> polyP / tryptase and <b>(C)</b> polyP / CD68 staining in sections of colonic adenocarcinoma specimens. <b>(B and D)</b> Data from six independent experiments presented as mean ± SD. Blue: PI, Green: (A and C) JC-D8 polyP-specific fluorescent probe, Red: (A) mast cell tryptase or (C) CD68. (A and C) One representative out of ten independent experiments is shown. Original magnification x600. Scale bar– 5μm. *P < 0.05, **P < 0.001, NS–not significant, HG–high grade.</p