Three different annealed silencer pre-designed siRNA oligonucleotides targeting the ptpn2 gene were obtained from Life Technologies Ltd. Per transfection, 100 pmol of each of the three gene specific siRNA oligonucleotides were transfected selleck chemicals Vandetanib into THP-1 cells using the Amaxa nucleofector system (Lonza, Walkersville, MD) according to the manufacturer��s instructions resulting in a final siRNA concentration of 1 nMol/ml. The achieved PTPN2 knock.-down was about 70%. After transfection, THP-1 cells were cultured in a 24-well plate for 36 h before treatment. Non-specific control siRNA (100 pmol/transfection, Life Technologies Ltd) was used as negative control. Enzyme-linked Immunosorbent Assay (ELISA) Supernatants from THP-1 cell cultures were collected and stored at ?80��C until further analysis.

ELISA kits for detection of human IL-6, and human monocyte chemoattractant protein (MCP)-1 were obtained from R&D Systems (Minneapolis, MN). Assays were performed according to the manufacturer��s instructions. Absorbance at 450 nm was determined using a BioTek Synergy 2 Microplate reader with Gen 5 Software version 5.1.11 (BioTek Instruments, Inc., Winooski, VT). Measurements were performed in duplicates. Statistical Analysis Data are presented as means �� standard deviations for a series of n experiments. Statistical analysis was performed by analysis of variance (ANOVA) followed by the Student�CNewman�CKeuls (for cell-line based experiments) or Mann-Whitney U (for mouse experiments) post hoc test. P values <0.05 were considered significant.

Results Spermidine Treatment Induces PTPN2 Protein Expression in Human Monocytic THP-1 Cells Because PTPN2 is crucially involved in negatively regulating IFN-�� induced signaling [17], we hypothesize that continuous pharmacological activation of PTPN2 by spermidine could attenuate the pro-inflammatory signaling induced by this cytokine. We thus investigated if PTPN2 activity and/or expression can be enhanced by spermidine in THP-1 monocytes and if this would attenuate the pro-inflammatory response to IFN-�� in these cells. To address the effect of spermidine on PTPN2 expression, THP-1 cells were incubated in the presence of IFN-�� and/or spermidine for 30 min or 36 h, and lysates were analyzed by Western blot using a monoclonal antibody specific for PTPN2. As shown in Figure 1a, neither IFN-�� nor spermidine treatment resulted in an increase in PTPN2 protein levels after 30 min.

However, there was a significant increase in PTPN2 levels in both IFN-�� or spermidine treated cells after 36 h treatment, but co-stimulation had no additive effect (Figure 1b). Figure 1 Effect of spermidine and interferon-gamma (IFN-��) treatment on protein tyrosine phosphatases (PTPN2 and Dacomitinib PTP1B) expression in human monocytic THP-1 cells.