MageA6 expression increases MageA11 protein levels.

<p>(A)Western blot showing Flag-MageA11 when co-expressed with increasing quantities of HA-MageA6 (0, 250, 500, 1000 and 1500 ng). GFP expression is the internal control. Membrane was probed with the indicated antibodies. (B) Western blot showing Flag-MageA11 when co-expressed with increasing quantities of HA-MHD-MageA6 (0, 250, 500, 1000 and 1500 ng). GFP expression is the internal control. Membrane was probed with the indicated antibodies. (C) Western blot showing Flag-MageA11 when co-expressed with increasing quantities of HA-MageA2 (0, 250, 500, 1000 and 1500 ng). GFP expression is the internal control. Membrane was probed with the indicated antibodies. (D) Western blot of LNCaP cells silenced (siA6/2) or not (siC) for MageA6 expression. Anti-pan MAGE-A antibody (6C1, Santa Cruz) was used to detect Mage-A proteins. 65KDa band corresponds to MageA11 while 45KDa band could correspond to different Mage-A proteins. GAPDH was used as loading control. (E)Left panel: Western Blot showing the endogenous levels of MageA11 in LNCaP stably expressing MageA6 (A6) or empty vector (EV). Extracts of HEK293T cells transfected with Flag-MageA11 (F-A11), HA-MageA6 (HA-A6) or empty vector (EV) were used as controls. MAGE-A detection was performed with anti-pan MAGE antibody (6C1, Santa Cruz). 65KDa band corresponds to MageA11 while 45KDa band could correspond to different Mage-A proteins. The observed increment in 45KDa band in LNCaP-A6 is caused by MageA6 stable expression. Right panel: quantification of MAGE-A11 vs β-tubulin band intensity corresponding to Fig 4E, lanes 4 and 5. (F) RT-qPCR for the determination of MageA11 mRNA levels in LNCaP-A6 (A6) and LNCaP-EV (EV). MageA11 mRNA was normalized to GAPDH mRNA levels. (G) RT-qPCR for the determination of PSA mRNA levels in LNCaP-A6 (A6) and LNCaP-EV (EV). PSA mRNA was normalized to GAPDH mRNA levels. Error bars indicate mean S.D. Student’s t test was used for statistical analysis. ** p < 0.001. * unspecific band. Triangles show the corresponding protein band and dashes mark the MW.</p

MageA6 and MageA11 co-expression in prostate cancer.

<p>Analysis of cBioPortal Cancer Genomics data sets form Prostate Adenocarcinoma and Testicular Germ Cell Cancer (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178370#sec002" target="_blank">Material and Methods</a>) as indicated in the two main columns. Gene overexpression was calculated by Z-score, defined as the relative expression of an individual gene to the gene’s expression distribution in a reference population. The indicated percent of over-expression refers to the number of samples over-expressing a given gene over the total of samples. Dot-plot graphics shows the correlation between MageA6 and MageA11 gene expression. Insets indicate Pearson and Spearman correlation scores.</p

Additional file 5: Table S4. of International core outcome set for clinical trials of medication review in multi-morbid older patients with polypharmacy

Specific decisions during the consensus meetings after Round 3. The table gives the elements of discussion and consensus obtained for six outcomes for which discrepancies were observed between stakeholder groups in the Delphi survey or for which a feasibility issue was raised by the participants. (PDF 77 kb

MageA6 enhances MageA11-dependent AR transcriptional activity.

<p>(A) Reporter gene assay for GR, MR and AR activity using specific gene-reporter in the presence or absence of MageA6 or MageA11 expression. Cells were treated with dexamethasone (Dx), Aldosterone (Aldo) or dihidrotestosterone (DHT) for 24 h prior to harvesting. The assay was performed in HEK293T cells. ev, empty vector. (B) Similar to A but combining MageA11 and MageA6 expression as indicated. (C) Determination of PSA mRNA levels through RT-qPCR. LNCaP cells were transfected with a siRNA control (siC) or a siRNA to silence MageA6 expression (siA6/2). DHT was added to cells as indicated. PSA mRNA levels were normalized to GAPDH mRNA levels. Error bars indicate mean S.D. Student’s t test was used for statistical analysis. * p < 0.05. ** p < 0.001.</p

Proteasome-dependent increase of MageA11 by MageA6 expression.

<p>(A)Western blot showing Flag-MageA11 alone or when co-expressed with HA-MageA6 after 0, 3 and 6 hours of cycloheximide (CHX) treatment. The membrane was probed with the indicated antibodies. (B) Western blot showing Flag-MageA11 expression in the presence of MG132 (1,5uM for 20h) or absence of MG132 (DMSO) after 0, 2, 4 and 6 hours of cycloheximide (CHX) treatment. The membrane was probed with the indicated antibodies. (C) Western blot showing Flag-MageA11 levels alone or when co-expressed with HA-MageA6 in the presence of MG132 or absence of MG132 (DMSO). GFP expression is the internal control. Membrane was probed with the indicated antibodies. *Unspecific band. Triangles show the corresponding protein band and dashes mark the MW.</p

Additional file 1: Table S1. of International core outcome set for clinical trials of medication review in multi-morbid older patients with polypharmacy

COS-STAR checklist as recommended by Kirkham et al. [30]. (PDF 162 kb

Additional file 4: Table S3. of International core outcome set for clinical trials of medication review in multi-morbid older patients with polypharmacy

Characteristics of the participants in the Delphi survey (all three rounds). (PDF 241 kb

Additional file 3: Table S2. of International core outcome set for clinical trials of medication review in multi-morbid older patients with polypharmacy

Names of the health domains and subdomains used to classify the outcomes extracted from the systematic review and the qualitative study, according to the OMERAT Filter 2.0 classification. (PDF 96 kb

Additional file 2: of International core outcome set for clinical trials of medication review in multi-morbid older patients with polypharmacy

Additional references: References of the 47 published studies and 32 RCT protocols identified in the systematic review. (PDF 107 kb

GTSE1 expression in breast cancer tumors and cells correlates with time to metastasis and invasiveness.

<p>(A) Kaplan–Meier survival curve of time to distant metastasis of breast cancer patients classified according to the expression of GTSE1. Red line: cases with high expression of GTSE1, blue line: cases with low expression of GTSE1. (p-value <10?–15) (B) Boxplots of the distribution of gene expression intensities of GTSE1 across different breast cancer subtypes (Grade 1, 2 or 3; p<10?-5; linear regression analysis),. (C) Western blot analysis of GTSE1 and EB1 protein levels in different breast cancer cell lines. Tumor types are: F, fibrocystic disease, non-transformed, immortal cell line; IDC, invasive ductal carcinoma; AC, adenocarcinoma; MC, metaplastic carcinoma. Invasive potential is characterized as not invasive (−), invasive (+), or highly invasive (++). Adapted from Neve et al. Cancer Cell 2006. (D) Quantitative RT-PCR analysis of GTSE1 and EB1 relative mRNA levels in MCF7 and MDA-MB-231 cells. Error bars represent the standard error of the mean from three independent experiments. p<0.01 (Student’s t-test). (E) Transwell migration assay and western blot of the MDA-MB-231 cell line. Cells were transfected with control (CON) or GTSE1 siRNA for 36 hours, trypsinized, and seeded on transwell membranes. Histograms show the mean number of cells/area that migrated through the transwell after 16 h (10 replicates/experiment). Error bars represent the standard error of the mean from three independent experiments. * indicates p<0.05 (Student’s t-test). Western blots were performed on cells after the same treatment, and blotted with anti-GTSE1 and anti-actin. (F) Transwell migration assay and western blot of the MCF7 cell line containing a stably integrated GTSE1 overexpression construct (pBABE-GTSE1) or empty vector (pBABE). Cells were trypsinized and seeded on transwell membranes. Histograms show the mean number of cells/area that migrated through the transwell after 16 h (10 replicates/experiment). Western blots were performed on cells after the same treatment, and blotted with anti-GTSE1 and anti-actin.</p