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Additional file 8: Figure 6. (a) Clonogenic agarose assay of HCC1806 cells transfected with siCtrl or siGYS1, cultured in 5.6 mM glucose complete medium for 10 days in normoxia. (b) Ki67 proliferation index in spheroids of HCC1806 cells transfected with siRNA-mediated GYS1 knockdown or scrambled control. (c) Well confluency of HCC1806-shCtrl and -shGYS1 cells treated with different concentrations of GTPP, cultured in 5.6 mM glucose complete DMEM, was measured by Incucyte every 3h.

Additional file 3: Figure S1. METABRIC mRNA expression data (27) and patient overall survival. (a) Overall survival of breast cancer patients with high or low mRNA expression of the respective glycogen enzymes in their primary tumor. Curves are separated by the median mRNA expression. (b) Overall survival curves of triple-negative patients only. (c) Overall survival of respectively ER-/HER2+, ER+/HER2+ or ER+/HER2- breast cancer patients with high or low GYS1 mRNA expression in their primary tumor. Curves are separated by the median GYS1 expression. (d) Overall survival multivariate Cox regression analyses correcting for Nottingham Prognostic Index (NPI) for all (left) and triple-negative breast cancer patients (right) with respectively high and low GYS1 mRNA in their primary tumors. GBE1 = glycogen branching enzyme 1; GYS = glycogen synthase; PYGB = brain glycogen phosphorylase; PYGL = liver glycogen phosphorylase; PYGM = muscle glycogen phosphorylase

Additional file 6: Figure 4. (a) Western blot of GYS1 demonstrates differential expression in breast cancer cell lines. (b) Knock-down of GYS1 in a broad panel of breast cancer cell lines, cultured in 5.6 mM glucose complete DMEM medium for 5 days, reduces cell growth in most breast cancer cell lines.

Additional file 5: Figure S3. Patient selection, survival curves and scoring examples of the primary breast tumor tissue micro-array. (a) Flowchart of the included tumor samples. (b) Corresponding PAS, PAS+diastase and CA9 staining of the areas depicted in Figure 1c. (c) Representative examples of staining intensity per scoring intensity and corresponding output of the analysis app in tumor cores and positive controls. (d) Additional boxplots of GYS1 and glycogen tumor H-score stratified by subtypes and Ki67 low vs. high subgroups within subtypes (top), and scores per tumor grade (bottom). (e) Heatmap of individual primary breast tumor samples (one sample per column) after unsupervised hierarchical clustering based on GYS1, glycogen and Ki67 tumor scores. White boxes in the annotation bars indicate missing values. (f) Overall survival curves of all patients included in the TMA analysis, stratified by respectively GYS1 or glycogen tertiles. Patients with more than one primary breast tumor (n = 9) were excluded from survival analyses.

Additional file 7: Figure 5. (a) Western blot confirmation of siRNA GYS1 knockdown, belonging to Figure 2c. (b) Clonogenic agarose assay of MDA-MB-231 cells with siCtrl or siGYS1, cultured in 5.6 mM glucose complete medium + 0.3% agarose for 14 days in normoxia. (c) Background intracellular glucose levels of MDA-MB-231 with scrambled control or GYS1 shRNA, after 48h culture in 5.6 mM glucose complete DMEM. (d) Proliferation of MDA-MB-231 cells with scrambled control or GYS1 shRNA, after 5 days culture in 10 mM complete DMEM.

Additional file 4: Figure S2. METABRIC mRNA expression data and patient survival. (a) Spearman correlations among GYS1 mRNA and GYS isoform 2 (GYS2), glycogen branching enzyme 1 (GBE1), glycogen breakdown enzymes glycogen phosphorylase muscle isoform (PYGM), brain isoform (PYGB) and liver isoform (PYGL), the hypoxic marker carbonic anhydrase 9 (CA9), and Marker of Proliferation Ki67 (MKI67). Numbers indicate Spearman’s p, only significant correlations are displayed. (b) GYS1 mRNA log2 expression levels across breast cancer clinical subtypes (left panel), tumor stage (middle) and tumor grade (right). Boxes represent the interquartile range and median, whiskers 1.5*interquartile range. Only significant Wilcoxon P-values are annotated. *P

Additional file 2: Table S2. Antibody and RNA specifications.

While initially subject to debate, meta-analyses have shown that eye movement desensitization and reprocessing (EMDR) is effective in the treatment of posttraumatic stress disorder (PTSD). Earlier studies showed that eye movements during retrieval of emotional memories reduce their vividness and emotionality, which may be due to both tasks competing for limited working memory (WM) resources. This study examined whether another secondary task that taxes WM has beneficial effects, and whether the stronger the taxing, the stronger the reductions in vividness/adversity. A reaction time (RT) paradigm showed that counting backwards requires WM resources, and that more complex counting is more demanding than simple counting. Relative to a retrieval-only condition, counting during retrieval of emotional memories reduced vividness and emotionality during later recall of these memories. However, the counting conditions did not differ in the magnitude of this reduction, and did not show the predicted dose-response relationship. Implications for a working-memory explanation of EMDR and for clinical practice are discussed.

Active Heymann nephritis in the rat is a model of idiopathic membranous glomerulopathy in man. The autoimmune response is directed to gp330, a large epithelial glycoprotein that is expressed on the tubular and the glomerular epithelium. Characteristic of the disease is the presence of immune complexes and complement in the glomerulus and proteinuria. We studied the effect of a new xenobiotic immunosuppressive agent, mycophenolate mofetil, on active Heymann nephritis. Mycophenolate mofetil significantly reduced the production of autoantibodies against gp330 in rats with Heymann nephritis. Glomerular deposition of IgG was not significantly lower in the treated groups than in the untreated groups with active Heymann nephritis, as detected by immunofluorescence staining. Glomerular complement component C3, however, was significantly lower in the mycophenolate mofetil treated rats. Treatment did not completely prevent the disease, but the percentage of rats that developed proteinuria in the treated groups was significantly lower than in untreated Heymann rats. The results of this study show that mycophenolate mofetil influences the T-cell-mediated humoral autoimmune response in active Heymann nephritis and results in a decreased severity of the disease.

The risk of diabetic nephropathy is partially genetically determined. Diabetic nephropathy is linked to a gene locus on chromosome 18q22.3-q23. We aimed to identify the causative gene on chromosome 18 and to study the mechanism by which the product of this gene could be involved in the development of diabetic nephropathy. DNA polymorphisms were determined in 135 case (diabetic nephropathy) and 107 control (diabetes without nephropathy) subjects. The effect of carnosine on the production of extracellular matrix components and transforming growth factor-beta (TGF-beta) after exposure to 5 and 25 mmol/l D-glucose was studied in cultured human podocytes and mesangial cells, respectively. A trinucleotide repeat in exon 2 of the CNDP1 gene, coding for a leucine repeat in the leader peptide of the carnosinase-1 precursor, was associated with nephropathy. The shortest allelic form (CNDP1 Mannheim) was more common in the absence of nephropathy (P = 0.0028, odds ratio 2.56 [95% CI 1.36-4.84]) and was associated with lower serum carnosinase levels. Car- nosine inhibited the increased production of fibronectin and collagen type VI in podocytes and the increased production of TGF-beta in mesangial cells induced by 25 mmol/l glucose. Diabetic patients with the CNDP1 Mannheim variant are less susceptible for nephropathy. Carnosine protects against the adverse effects of high glucose levels on renal cells.