In contrast to the observed increase in PTPN2 expression, neither IFN-�� nor spermidine treatment changed the expression levels of PTP1B (Figures 1c and 1d). Taken together, this indicates that both IFN-�� and spermidine specifically induce PTPN2 expression after 36 h treatment. Spermidine Treatment selleck Increases Phosphatase Activity of PTPN2 in Human Monocytic THP-1 Cells To further explore the effects of spermidine on PTPN2 in human THP-1 cells, we measured the phosphatase activity of PTPN2 in response to treatment with IFN-�� and/or spermidine by performing immunoprecipitation of PTPN2 from whole cell lysates and analyzing its phosphatase activity using a fluorescence-based assay. As shown in Figure 2a, a significant increase in PTPN2 phosphatase activity was observed after treatment of cells for 30 min with spermidine or with IFN-�� and spermidine (p<0.

01 and p<0.001, respectively), compared to untreated cells or IFN-��-treated cells. This indicates that spermidine increases enzymatic activity of PTPN2 at a time point in which protein expression is not affected (Figure 1a). However, at this time point co-treatment with spermidine and IFN-�� did not have a statistically significant additive effect on PTPN2 activity, compared to treatment with spermidine or IFN-�� alone. When cells were incubated for 36 h, IFN-�� alone was able to significantly increase the phosphatase activity of PTPN2 (p<0.001; Figure 2b), which is consistent with our previous results [18]. Moreover, cells treated with spermidine alone showed significantly increased PTPN2 activity at 36 h, with nearly a 3-fold induction compared to untreated cells (p<0.

001, Figure 2b). The co-treatment of THP-1 cells with both IFN-�� and spermidine for 36 h significantly further increased PTPN2 phosphatase activity, compared to either IFN-�� or spermidine alone (p<0.01 and p<0.05, respectively; Figure 2b). This observation clearly indicates an additive effect of both stimuli at this time point with respect to PTPN2 phosphatase activity, effect that was not observed on protein expression (Figure 1b). Because phosphatase activity was normalized to the PTPN2 protein content of the cells, the observed increase in activity is not due to the increase in PTPN2 protein level, but rather to actual activation of the available PTPN2 protein, and appears to occur independently of the increase in PTPN2 expression levels. Figure 2 Protein tyrosine phosphatase non-receptor type 2 (PTPN2) phosphatase Entinostat activity in interferon-gamma (IFN-��)- and/or spermidine-treated human monocytic THP-1 cells.