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Expression of CCKBR in gastric adenocarcinoma cells. AGS cells have low abundance of the CCKBR, MKN45 express the CCKBR endogenously and AGS-Gr cells are stably transfected with the CCKBR. S1b. Negative control images of the MKN45 cells stained for the CCKBR (phase contrast, Alexa 488, Draq5). Figure S2: Gastrin induces autophagy. AGS-Gr (a & b) cells treated with gastrin (10 nM), BafA1 (100 nM) and gastrin + BafA1 for 2 and 4 h. Protein expression of MAP1LC3B-II and SQSTM1 was analyzed by immunoblotting. The images shown represent one of three independent experiments. Graphs show mean +/- SEM (P- values: *** ≤ 0.01 **≤ 0.02, and * ≤ 0.05). Figure S3: Negative controls (primary antibodies omitted) for MAP1LC3B (Alexa 488) and SQSTM1 (Alexa 647). Figure S4: Gastrin mediated survival is dependent on autophagy. (a): A representative cytometric plots showing AGS-Gr cells treated with BafA1 and gastrin for 18 h. Cell viability was assessed using annexin V-PI staining and flow cytometric analyses. Blocking autophagy reduces gastrin mediated survival in AGS-Gr cells. (b): Cell viability assessed in AGS-Gr cells treated with gastrin (10 nM) for 6- 72 h. (c & d): Cells treated with gastrin (2 h) and subsequently treated with increasing concentrations of cisplatin. Viability was assessed at 24 and 72 h. Results show mean +/-SD (n=3, P-values: * ≤ 0.05 ** ≤ 0.01 *** ≤ 0.001). (e): AGS-Gr cells treated with HCQ for 8 h. Protein expression of MAP1LC3B-II and SQSTM1 was detected by immunoblotting. (f) Gastrin induced autophagy is dependent on ULK1: AGS-Gr cells treated with gastrin, BafA1 and ULK1 inhibitor SBI-0206965 (10 μM) for 4 h. Protein expression of MAP1LC3B-II and SQSTM1 was detected by immunoblotting. The immunoblots represent one of three independent experiments. Figure S5: Inhibition of gastrin induced autophagy by Comp C. AGS-Gr cells pretreated with Compound C (10 μM) for 12 h before adding BafA1 and gastrin (4 h). Protein expression of SQSTM1 is shown by immunoblotting. The blot represents one of two independent experiments. (DOCX 2504 kb)

Tumour stiffness is increasingly seen as a clinically relevant parameter, with increased stiffness being linked to poorer prognosis, but it is also one that is not inherently easy to measure either in vivo or ex vivo. We have established a testing protocol for fresh ex vivo tumour tissue from tumour bearing mice using a Stable Micro Systems texture analyser fitted with a 2mm diameter cylindrical probe. The data demonstrate that, in this tumour model, changes in tumour growth time alter the tumour stiffness, but the presence or absence of T-cells does not significantly alter tumour stiffness. However, there may be more substantial differences if a more immunogenic tumour model was employed. (c) the author

Kimura's disease is an uncommon entity that affects adults, with a predilection for the Asian population. This may rarely be encountered in children, and the knowledge of this fact is essential to rule out the remote possibility of Kimura's disease in children with a slow-growing painless mass in the head and neck region. In this case report, we document this disease in an 8-year-old boy with a slow-growing swelling in the right posterior auricular region.

The peptide hormone gastrin is known to play a role in differentiation, growth and apoptosis of cells in the gastric mucosa. In this study we demonstrate that gastrin induces Nuclear Receptor 4A2 (NR4A2) expression in the adenocarcinoma cell lines AR42J and AGS-GR, which both possess the gastrin/CCK2 receptor. In vivo, NR4A2 is strongly expressed in the gastrin responsive neuroendocrine ECL cells in normal mucosa, whereas gastric adenocarcinoma tissue reveals a more diffuse and variable expression in tumor cells. We show that NR4A2 is a primary early transient gastrin induced gene in adenocarcinoma cell lines, and that NR4A2 expression is negatively regulated by inducible cAMP early repressor (ICER) and zinc finger protein 36, C3H1 type-like 1 (Zfp36l1), suggesting that these gastrin regulated proteins exert a negative feedback control of NR4A2 activated responses. FRAP analyses indicate that gastrin also modifies the nucleus-cytosol shuttling of NR4A2, with more NR4A2 localized to cytoplasm upon gastrin treatment. Knock-down experiments with siRNA targeting NR4A2 increase migration of gastrin treated adenocarcinoma AGS-GR cells, while ectopically expressed NR4A2 increases apoptosis and hampers gastrin induced invasion, indicating a tumor suppressor function of NR4A2. Collectively, our results uncover a role of NR4A2 in gastric adenocarcinoma cells, and suggest that both the level and the localization of NR4A2 protein are of importance regarding the cellular responses of these cells. Copyright: © 2013 Misund et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Using genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens to understand endocrine drug resistance, we discovered ARID1A and other SWI/SNF complex components as the factors most critically required for response to two classes of estrogen receptor-alpha (ER) antagonists. In this context, SWI/SNF-specific gene deletion resulted in drug resistance. Unexpectedly, ARID1A was also the top candidate in regard to response to the bromodomain and extraterminal domain inhibitor JQ1, but in the opposite direction, with loss of ARID1A sensitizing breast cancer cells to bromodomain and extraterminal domain inhibition. We show that ARID1A is a repressor that binds chromatin at ER cis-regulatory elements. However, ARID1A elicits repressive activity in an enhancer-specific, but forkhead box A1-dependent and active, ER-independent manner. Deletion of ARID1A resulted in loss of histone deacetylase 1 binding, increased histone 4 lysine acetylation and subsequent BRD4-driven transcription and growth. ARID1A mutations are more frequent in treatment-resistant disease, and our findings provide mechanistic insight into this process while revealing rational treatment strategies for these patients.