Irradiating the lungs of young adult female FVB/N mice with either a single 12 Gy dose or fractionated 24 or 30 Gy doses of ¹³⁷Cs γ-rays leads to a marked decrease in the expression of aquaporin-5 in the lung.

<p>Lung tissues were collected at 14 weeks (12 Gy and 30 Gy) or at 1, 2 and 5 months (24 Gy) PI. Cell lysates were generated from the snap-frozen left lung from 4 mice and run on a 10–15% polyacrylamide mini-gel. Each lane contains the pooled lysate from 2 mice. ß-actin served as loading control. <b>A</b>: The reduction in aquaporin-5 protein at 14 weeks after a single 12 Gy or a fractionated 30 Gy dose is similar. <b>B</b>: The reduction in aquaporin-5 protein occurs as early as 1 month after a fractionated 24 Gy dose and remains reduced for the next 4 months. <b>C</b>: Quantification of the aquaporin-5 protein levels in 3B shows that the reduction in aquaporin-5 is progressive over the first 2 months postirradiation (PI) and then remains at a relatively constant low level up to 5 months PI. Densitometry was used to quantify the protein in each lane; all irradiation values in 2C were normalized to the unirradiated control values obtained on the same gel. The data in 2C are the mean ± 1 SEM.</p

Irradiating the lungs of young adult female FVB/N mice with either a single 12 Gy dose or fractionated 24 or 30 Gy doses of ¹³⁷Cs γ-rays leads to decreased levels of E-cadherin and increased levels of vimentin protein in the lung.

<p><b>A, C</b>: Although the vimentin data is variable, the decrease in E-cadherin and the increase in vimentin are similar at 14 weeks after a single 12 Gy dose or a fractionated 30 Gy dose. <b>B, D</b>: After a fractionated 24 Gy dose, there is a slight increase in E-cadherin at 1 month postirradiation (PI) followed by a progressive decrease up to 5 months PI. After a fractionated 24 Gy dose, there is a progressive increase in vimentin up to 2 month PI that remains relatively constant for the next 3 months. All methods and analyses are identical to those described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053628#pone-0053628-g003" target="_blank">Fig. 3</a>.</p

Irradiating the lungs of young adult female FVB/N mice with single and fractionated doses of ¹³⁷Cs γ-rays leads to dose-dependent increases in mortality.

<p>Groups of 8–10 week-old female FVB/N mice were irradiated with single (11–13 Gy) or fractionated (18–36 Gy) doses of γ rays, and their survival recorded up to 22 weeks postirradiation.</p

Irradiating the lungs of young adult female FVB/N mice with either a single 12 Gy dose or fractionated 24 or 30 Gy doses of ¹³⁷Cs γ-rays leads to increase in VCAM-1 and TGF-ß1 protein in the lung.

<p><b>A</b>: The increase in VCAM-1 and TGF-ß1 is similar at 14 weeks after a single 12 Gy dose or a fractionated 30 Gy dose. <b>B</b>: Both VCAM-1 and TGF-ß1 increase up to 2 months after a fractionated 24 Gy dose and then decrease up to 5 months postirradiation.</p

Irradiating the lungs of young adult female FVB/N mice with single and fractionated doses leads to regional fibrosis and collagen deposition and increased inflammatory cell infiltration.

<p>Female FVB/N mice were received thoracic radiation with 12 and 13 Gy single dose or fractionated dose 24 Gy. Mice were euthanized at 14 weeks (12 and 13 Gy groups) or 1, 2 and 5 months (24 Gy fractionated group) postirradiation, respectively. Lung sections were stained with H & E, Massons Trichrome and/or α-SMA. A shows representative lung images of H & E staining (control: a and d; irradiated with a single 12 Gy: b and e; 13 Gy: c and f) and Masson's trichrome staining (control: g; 12 Gy: h) for mouse treated with a single dose radiation. B shows representative lung images of H & E staining (control: a and d; 1 m postirradiation (PI) with 24 Gy fWTI: b and f; 2 m PI: c and g; 5 m PI: d and h) and α-SMA staining (control: i; 1 m PI: j; 2 m PI: k; 5 m PI: l) for mice treated with 24 Gy dose of fWTI.</p

Pro-SP-c and α-SMA protein co-localize in the lungs of FVB/N mice at 14 weeks postirradiation with a single dose of 12 Gy.

<p>Lung section (5 µm) from an unirradiated control (A) and an irradiated mouse (B) were co-stained with pro-SP-c and α-SMA antibodies. In the representative photomicrographs the pro-SP-c protein is red, the α-SMA protein is green, and the nuclei are blue.</p

Irradiating the lungs of young adult female FVB/N mice with either a single 12 Gy dose or fractionated 24 or 30 Gy doses of ¹³⁷Cs γ-rays leads to increases in pro-SP-c protein and pro-SP-c positive cells in the lung.

<p><b>A</b>: The increase in pro-SP-c⁺ cells is similar at 14 weeks after a single 12 Gy dose or a fractionated 30 Gy dose. <b>B</b>: The increase in pro-SP-c⁺ cells after a fractionated 24 Gy dose is progressive over the first 2 months postirradiation (PI) and then remains relatively constant up to 5 months PI. <b>C</b>: Upper gel, the increase in pro-SP-c protein at 14 weeks after a single 12 Gy or a fractionated 30 Gy dose is similar. Lower gel, pro-SP-c protein is increase by 1 month PI and remains relatively constant up to 5 months PI. For 4A and 4B, lung tissues were collected and prepared for histochemical analysis either at 14 weeks (12 Gy and 30 Gy) or 1, 2 and 5 months (24 Gy) PI. Sections (5 µm) of lung tissue were stained with pro-SP-c antibodies, and the pro-SP-c⁺ cells were analyzed using morphometric method. Data are the mean ± 1 SEM; n = 3; ^*<i>p</i><0.05 vs. sham control. The gels in 4C were prepared and analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053628#pone-0053628-g003" target="_blank">Fig. 3</a>.</p

Additional file 2: Figure S1. of Circulating mutational portrait of cancer: manifestation of aggressive clonal events in both early and late stages

ctDNA mutational landscape for major cancer groups for the top 30 genes in total cohort. Figure S2. ctDNA mutational landscape of patients’ stage known for the top 30 genes in lung cancer cohort. Figure S3. Higher mutation numbers in the ctDNA are associated with decreased survival in lung cancers. Higher mutation numbers in ctDNA are associated with poor survival. “n” defines the number of mutations and survival plots are separated by mutation numbers: n = 1, 2, 3, 4, 5, and 6 mutations. Blue lines indicate more than “n” mutations and the pink lines indicate equal to or less than “n” mutations. P-values were derived using the log-rank test. Figure S4. The concordance of top mutated genes in the ctDNA and tDNA of 37 lung cancer patients. (DOC 1098 kb

Additional file 1: Table S1. of Circulating mutational portrait of cancer: manifestation of aggressive clonal events in both early and late stages

Clinical and treatment characteristics of the 18 cases with no ctDNA alterations detected. Table S2. Description of mutation load in total cohort (n = 177). Table S3. All mutations detected in patient ctDNA compared with other studies. (DOC 625 kb