Additional file 1: of Expression of the glucagon-like peptide-1 receptor and its role in regulating autophagy in endometrial cancer

Table S1. Detailed data of participants. Clinicopathological data of 154 patients with endometrial cancer who underwent surgery at the Teikyo University Hospital. These clinical characteristics were obtained by a retrospective review of medical records and the pathological data were obtained by our tissue microarray. (XLSX 14 kb

Inhibition of cell proliferation and augmentation of G1 arrest by combination of a MEK inhibitor and NVP-BEZ235 (or RAD001) in cells with alterations in

<p><b><i>K-Ras</i></b><b> (mutation or amplification).</b> (A)–(B) WST-8 assay was performed in HEC-1B (group C) and HEC-50B (group D) cell lines. All the experiments were repeated three times and each value is shown as the mean of three experiments +/− S.D. The combination of a MEK inhibitor (PD98059 or UO126) and NVP-BEZ235 (or RAD001) significantly augmented anti-proliferative effect in these cell lines, compared with either inhibition alone (p<0.05 by Student's t-test). (C)–(D) Flowcytometric analysis of cell cycle in HEC-1B and HEC-50B cells. All experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± S.D. Combination of a MEK inhibitor (PD98059 or UO126) and NVP-BEZ235 significantly augmented the percentage of cells in the G0-G1phase of the cell cycle in these cells. *: p<0.05 by Student's t-test.</p

in vivo anti-tumor effect of NVP-BEZ235 and RAD001 in nude mice.

<p>Subcutaneous xenograft tumors in athymic BALB/c mice were established in the injection of endometrial carcinoma cells. Mice were treated with a daily dose of 40 mg/kg (NVP-BEZ235) or with a daily dose of 2.5 mg/kg RAD001 or vehicle alone (control). Each treatment group contained 6 mice; (A) HEC-59 and (B) AN3CA. Tumor volumes were calculated by the formula {(major axis)*(minor axis)2/2}mm3 twice a week. Groups were compared at the end of treatment. Points, mean; bars, SD, *;p<0.05. (C) Appearance of subcutaneous tumors in HEC-59 xenografts. (D) Western blot of total lysates from the HEC-59 xenografts. p-Akt, p-FOXO1/3a, p-GSK3beta, p-S6 were assessed 1 and 24 h after the last drug administration. Total Akt and beta-actin were shown as loading controls.</p

Copy number gain at the locus of

<p><b><i>K-Ras</i></b><b>(12p12.1) in the two group D cell lines.</b> (A) SNP array ‘karyograms’ (250K) of HEC-50B cells. The graph shows the total copy number through chromosome 9p-X. The locus of <i>K-Ras</i> is amplified as indicated. (B) Array CGH of KLE cells. The graphs show total copy number throughout the entire genome (Left) and chromosome 12 (Right). The locus of <i>K-Ras</i> is amplified as indicated.</p

Inhibition of cell proliferation by NVP-BEZ235 and RAD001.

<p>(A)–(D) WST-8 assay showing the sensitivity of endometrial cancer cells to NVP-BEZ235 and RAD001 at increasing concentrations of drug (nmol/L) for 72 h. The data was normalized to the value of control cells. (A) Four group A cell lines with double mutations of <i>PIK3CA</i> and <i>PTEN</i>, (B) Five group B cell lines with <i>PTEN</i> mutations, (C) Two group C cell lines with coexistent mutations of <i>K-Ras</i> and <i>PIK3CA</i>, and (D) Two group D cell lines with chromosomal copy number amplification at the locus of <i>K-Ras</i>. All experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± S.D.</p

Flowcytometric analysis of cell cycle in cancer cells treated with either NVP-BEZ235 or RAD001.

<p>(A–D) Cells (5×10⁵) were seeded in the presence of 10% serum and treated with NVP-BEZ235 or RAD001 for 48 h at a dose of 10 nM or 100 nM, respectively. A higher dose of NVP-BEZ235 (100 nM) significantly augmented the percentage of cells in the G0-G1phase of the cell cycle, compared with that of RAD001 (100 nM). (A)–(B); The data from two group A cells. (C)–(D); the data from two group B cells.</p

Measurement of the signaling entropy in GSE63514.

<p>The entropy rate was significantly higher according to the disease progression (Kruskal-Wallis test, <i>p</i> < 0.0001). Cancer; invasive cancer.</p

Measurement of the signaling entropy in GSE27678.

<p>(A) Boxplot of the entropy rate from whole samples. The entropy rate increased according to the disease progression (Kruskal-Wallis test, <i>p</i> < 0. 001). Among them, the entropy rate from the cell line was highest, as described in the literature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176353#pone.0176353.ref009" target="_blank">9</a>]. (B) Entropy rate from each platform. ○ represents the data obtained from the Affymetrix Human Genome U133A 2.0 Array, and * represents the data obtained from the Affymetrix Human Genome U133 Plus 2.0 Array. The distribution patterns of the entropy rate seemed slightly different among the platforms.</p

Measurement of the signaling entropy in GSE75132.

<p>The median of the entropy rate was higher in the HPV16-persistent (HPV16+) groups than the HPV-negative (HPV-) group. The group that was destined to progress to CIN3+, or the HPV16-persistent with progression group, tended to have a higher entropy rate than the HPV16-persistent without progression group.</p

Dynamic Motion and Rearranged Molecular Shape of Heme in Myoglobin: Structural and Functional Consequences

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