Additional file 2: Supplemental Figure S1. of MicroRNA profiling of primary pulmonary enteric adenocarcinoma in members from the same family reveals some similarities to pancreatic adenocarcinoma—a step towards personalized therapy

Immunohistochemical analyses in the cytological sample of the proband’s brother. The stainings for TTF1, CK7, CDX-2, and CK20, summarized in the table, showed patterns of protein expression similar to the expression levels observed in the PEAC component of the proband’s sample. Left column (original magnification ×20); right column (original magnification ×40). (PPTX 32,747 kb

Additional file 3: Supplemental Figure S2. of MicroRNA profiling of primary pulmonary enteric adenocarcinoma in members from the same family reveals some similarities to pancreatic adenocarcinoma—a step towards personalized therapy

PCR analysis of miR-31*/-126*/-506/-508-3p/-514. Quantitative “miRNA-targeted” real-time PCR of five miRNA aberrantly expressed in the proband’s PEAC according to the microarray results showed that the microarray data were validated by PCR data as well as that the pattern of modulation of expression of these miRNA was similar in the proband’s brother. Columns and bars, average levels ± SD, compared using the 2-ΔΔCt method to a pool of normal lung tissues, dashed line. (PPTX 158 kb

Additional file 1: of MicroRNA profiling of primary pulmonary enteric adenocarcinoma in members from the same family reveals some similarities to pancreatic adenocarcinoma—a step towards personalized therapy

Analyses of EGFR, KRAS, ALK and MSI. Detailed methods used for the analyses of EGFR, KRAS mutation, ALK rearrangement, MSI, and miRNA-PCR. (DOC 108 kb

Down-regulation of nuclear expression of NF-kB p65 subunit LPS-stimulated BV2 microglial cells.

<p>BV2 cells were stimulated with 100 ng/ml LPS as described before. Both nuclear and cytoplasmic localizations of p65 ware evaluated using anti-p65 polyclonal antibody and a FITC-labelled anti-rabbit IgG antibody (Green color, lines 2). DAPI was used in order to identify nuclei (Blue color, line 1). Images of cells were obtained by bright field light (BF, line 4) and UV light excitation lines 1,2 and 3). Nuclear translocation of p65 subunit is visible by detection of Aqua color (Merge, line 3). Cytoplasmic p65 expression was associated with different intensity of green color inside the cytoplasm. To visualize better the Aqua color co-localized into the nuclei, we overlapped the BF images upon merge of fluorescence (line 5). The bars reported different combinations of treatments in BV2 cells as follows: CTRL (LPS-/TVE10%-), LPS (LPS+/TVE10%), TV (LPS-/TV10%+) and LPS+TV (LPS+/TV10%+). The clear Aqua color was present only in LPS treated cells. No Aqua color significant differences were observed comparing CTRL <i>vs</i> TV and LPS+TV colums.</p

<i>AKT1</i> and <i>SELP</i> Polymorphisms Predict the Risk of Developing Cachexia in Pancreatic Cancer Patients

<div><p>Pancreatic ductal adenocarcinoma (PDAC) patients have the highest risk of developing cachexia, which is a direct cause of reduced quality of life and shorter survival. Novel biomarkers to identify patients at risk of cachexia are needed and might have a substantial impact on clinical management. Here we investigated the prognostic value and association of <i>SELP-rs6136</i>, <i>IL6-rs1800796</i> and <i>AKT1-rs1130233</i> polymorphisms with cachexia in PDAC. Genotyping was performed in DNA from blood samples of a test and validation cohorts of 151 and 152 chemo-naive locally-advanced/metastatic PDAC patients, respectively. The association of <i>SELP-rs6136</i>, <i>IL6-rs1800796</i> and <i>AKT1-rs1130233</i> polymorphisms with cachexia as well as the correlation between cachexia and the candidate polymorphisms and overall survival were analyzed. Akt expression and phosphorylation in muscle biopsies were evaluated by specific ELISA assays. <i>SELP-rs6136-AA</i> and <i>AKT1-rs1130233-AA/GA</i> genotypes were associated with increased risk of developing cachexia in both cohorts (<i>SELP: p</i> = 0.011 and <i>p</i> = 0.045; <i>AKT1: p</i> = 0.004 and <i>p</i> = 0.019 for the first and second cohorts, respectively), while patients carrying <i>AKT1-rs1130233-GG</i> survived significantly longer (<i>p</i> = 0.002 and <i>p</i> = 0.004 for the first and second cohorts, respectively). In the multivariate analysis <i>AKT1-rs1130233-AA/GA</i> genotypes were significant predictors for shorter survival, with an increased risk of death of 1.7 (<i>p</i> = 0.002) and 1.6 (<i>p</i> = 0.004), in the first and second cohorts, respectively. This might be explained by the reduced phosphorylation of Akt1 in muscle biopsies from patients harboring <i>AKT1-rs1130233-AA/GA</i> (<i>p</i> = 0.003), favoring apoptosis induction. In conclusion, <i>SELP</i> and <i>AKT1</i> polymorphisms may play a role in the risk of cachexia and death in PDAC patients, and should be further evaluated in larger prospective studies.</p></div

Akt1 expression in muscle samples according to the <i>AKT1-rs1130233</i> polymorphism.

<p>Bar graphs illustrating the mean±SD expression of total Akt1 (<b>A</b>) and phospho-Akt1 (<b>B</b>) in muscle samples from patients with differential <i>AKT1-rs1130233</i> genotypes (N = 9 samples in each group) *<i>p</i><0.05.</p

Effect of TVE on NO, IL-6, PGE2 and TNFα release in BV2 cells.

<p>BV2 cells were exposed to TVE for 12 hours at different concentrations (1-2.5-5-10%). LPS was added 12 hours after TVE pre-incubations. The release was evaluated 24 hours after LPS exposure. Data were expressed as percentage of target release with respect to untreated cells (100%). N = 6 for each experimental group. * p<0.05 <i>vs</i> control non-treated cells; ** p<0.05 <i>vs</i> LPS treated cells. Graphs represented the results for the following markers: <b>A)</b> NO; <b>B)</b> IL-6; <b>C)</b> PGE2 and <b>D)</b> TNFα. All concentrations were expressed as (pg/mL).</p

Correlation between cachexia and candidate <i>SELP</i>, <i>AKT1</i> and <i>IL-6</i> SNPs.

<p><i>*p-values were calculated with Fisher's exact test.</i></p><p><i>SNPs, single nucleotide polymorphisms.</i></p><p>Note: <i>SELP</i> and <i>IL6</i> SNPs was detectable in all the samples, while the <i>AKT1</i> genotype could not be determined in 2 of the patients of the second cohort.</p><p>Correlation between cachexia and candidate <i>SELP</i>, <i>AKT1</i> and <i>IL-6</i> SNPs.</p

Clinical outcome according to cachexia and <i>AKT1-rs1130233</i> polymorphism.

<p>(<b>A</b> and <b>B</b>) Kaplan–Meier survival curves according to cachexia in the first and second cohort of patients. (<b>C</b> and <b>D</b>) Kaplan–Meier survival curves according to the <i>AKT1-rs1130233</i> polymorphism in the first and second cohort of patients. <i>p</i>-values were calculated with the log-rank test. OS: Overall survival.</p

Clinical outcome according to candidate <i>SELP</i>, <i>AKT1</i> and <i>IL-6</i> SNPs.

<p><i>OS: overall survival; mo: months.</i></p><p>Clinical outcome according to candidate <i>SELP</i>, <i>AKT1</i> and <i>IL-6</i> SNPs.</p