Here we show that the extracellular domain of Msb2 is shed in response to specific environmental cues

dge, UK). The secondary antibodies and isotype controls used for immunoblotting, immunohistochemistry, immunofluorescence, and FACS analyses are indicated in the respective sections. siRNA Transfection and Functional Studies To confirm the specificity of the anti-CSPG4 antibodies and to evaluate the functional relevance of pCSPG4 in pancreatic cancer, we used siRNA-based knock-downs. Cells were grown up to 50 70% confluence and transfected using the HiPerFect transfection reagent at 10 nM with duplex oligonucleotides: siRNA set 1, siRNA set 2 , or control siRNA set for 48 hours. To evaluate the effect of CSPG4 gene silencing on cell functions, proliferation, migration, and invasiveness, the cells were treated with control or CSPG4-specific siRNA and analyzed through MTT-based growth assay, scratch test, and Matrigel-based invasion assay, using the standard techniques reported elsewhere. Materials and Methods Serum and Tissue Sampling The analyses included the pancreatic biopsies and sera from donors and patients with chronic pancreatitis or different variants of exocrine pancreatic tumors: benign, premalignant and malignant. The study was approved by the Ethics Committee of the Faculty of Medicine, University of Heidelberg, Germany and performed with patients’ written informed consent and in compliance with institutional regulations. Freshly removed tissues were flash-frozen in liquid nitrogen for RNA and western blot profiling, or fixed in paraformaldehyde solution for 1224 h prior to paraffin embedding for histological analysis. Serum sCSPG4 was measured using ELISA in test and validation cohorts comprising donors and patients with chronic pancreatitis or tumors: i) benign, ii) premalignant and with high-grade dysplasia/carcinoma in situ ), and iii) malignant and ductal adenocarcinomas including anaplastic, adenosquamous and PDAC ). Pancreatic pCSPG4 expression was evaluated using qRT-PCR, western blot analysis and immunohistochemistry. The patients’ characteristics are given in Induction of Hypoxia Pancreatic cell lines were 22408714 grown up to 70% confluence, transferred to the modular incubator 16041400 chamber, flashed for 30 min with the hypoxic gas mixture, and incubated in the closed unit for 3 h or 48 h at 37uC. The same procedure was performed without exposure to hypoxic gas to obtain a normoxic control. FACS Analysis Pancreatic cell lines were suspended in FACS Buffer, blocked with FcR Blocking Reagent, and incubated for 20 min with mouse anti-CSPG4 antibody at room temperature, or IgG1 isotype control. We used directly labeled phycoerythrin -conjugate or unlabeled antibody with subsequently added anti-mouse AlexaFlour488-conjugate. Measurements of expression under normoxic and hypoxic conditions were performed using the FACScan and LSR flow cytometers. Cell Cultures, Media, Antibodies Nine DSMZ-certified pancreatic cancer cell lines and the cervical carcinoma HeLa cell line were cultured in RPMI medium supplemented with 10% fetal bovine serum. Primary pancreatic stellate cells were obtained through the outgrowth method of AG-221 Bachem et al., cultured in low glucose DMEM/F12 medium supplemented with 20% FBS and propagated for up to 8 passages as previously described. Immortalized human pancreatic ductal epithelial cells were received as a gift, and cultured in serum-free keratinocyte medium, supplemented with 5 ng/ml recombinant epidermal growth factor and 50 mg/ml bovine pituitary extract. The panel of primary antibodies included the mouse monoc

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