Figure 5.

Effect of PTP1B silencing on the response of human macrophages. Human monocytes from buffy coats were differentiated into macrophages and treated with a mixture of sc- or siRNA oligonucleotides to silence PTP1B. After incubation for 24 h with 200 ng/ml of LPS plus 20 ng/ml of human IFN-γ, 25 μg/ml of polyI:C or 20 ng/ml of each human IL-4+IL-13, the levels of PTP1B and COX2 were determined (a). The time course of the phosphorylation and total levels of proteins related to NF-κB, AKT and MAPKs were determined (b). The release of TNF-α and PGE₂ to the culture medium was determined with specific kits (c). The induction of apoptosis, measured as the annexin V-positive population, was determined in both resting and LPS+IFN-γ activated cells (d). In addition to this, to determine the sensitivity of cells silenced for PTP1B to apoptosis in response to pro-inflammatory dependent stimuli the time course and the sensitivity at 36 h to different concentrations of staurosporine (as an stressor to promote mitochondrial-dependent apoptosis) were determined (d). Results show a representative experiment or the mean±S.D. of three independent experiments. *P<0.05; **P<0.01 versus the same condition in cells treated with scRNA