3) (P < 0·05). MDR1 and MRP inhibitors induced a marked decrease in mDCs [half maximal inhibitory concentration (IC50): P-glycoprotein inhibition using valspodar (PSC833 5 μM, CAS 115104-28-4 (MK571) 50 μM and probenecid 2·5 μM] Selleck Tyrosine Kinase Inhibitor Library and an increase in iDCs. Thus, after hypoxia, PSC inhibited mDCS to 31·4% (P < 0·05), MK571 to 40% (P < 0·05) and PBN to 45·6% (P < 0·05). The effect of ABC blockers on DC maturation after LPS showed similar results: PSC833

reduced mDCS to 48·8% (P < 0·05), MK571 to 51·6% (P < 0·05) and PBN to 50·6% (P < 0·05). All mean values were analysed for 10 experiments. To rule out a toxic effect of inhibitors on DC viability, cell apoptosis was analysed by annexin V/7-ADD assay. A comparable percentage of viable cells was observed after hypoxia exposure with or without ABC inhibitors exposure (H: 86·1%, H + PSC: 84·25%, H + MK: 85·29% and H + PBN: 83·7%). We found no statistical mTOR inhibitor changes between hypoxia DC and non-stimulus. Hypoxic conditions induced a twofold

DC maturation compared to control non-stimulated DCs (P < 0·05), analysed as intensity of different maturation markers (CD40, CD83, HLADR and CD54). This confirmed the results validated in a previous study [8]. ABC inhibitors showed a clear decrease in both plamacytoid-like and conventional DC phenotype maturation, depending on the stimuli (Table 1). When iDCs were stimulated by LPS the mean fluorescence intensity (MFI) of CD80, CD86, HLA-DR and CD54 maturation markers increased MFI threefold with respect to control, and there was a twofold increase of MFI with respect to hypoxia stimulus (Table 1). Interestingly, CD83 and CD40 were similarly up-regulated under both stimuli, and CD86 was down-regulated under hypoxia-achieving control values, suggesting a plasmocytoid-like phenotype pattern with respect to LPS-DC. Despite these differences in the maturation response of DCs after the two stimuli, the up-regulation of maturation markers was abrogated strongly when ABC inhibitors were added at a similar intensity (Table 1). All results are representative of six experiments. Figure 4 is a representative histogram of the most relevant changes in DC maturation markers

Methisazone after hypoxia or LPS. HIF-1α expression in control cells was 37·5 ± 5·2%, when DCs stimulated by hypoxia were increased significantly with respect to control (67·6 ± 3·7). Interestingly, when ABC inhibitors were added to hypoxic-DC, HIF-1α results were similar to hypoxia-DCs (H + PSC833 57·5 ± 4·4 and H + MK571 62·3 ± 5·1) (Fig. 5). To address the functional impact of ABC transporter inhibition on DCs, we next assessed the effects of these cells on lymphocyte proliferation in the MLR, evaluated by CFSE staining. Hypoxia- and LPS-matured DCs were capable of inducing a significantly (P < 0·05) higher lymphocyte proliferation than non-stimulated iDCs. Functional studies showed a higher T cell proliferation after LPS than after hypoxia stimulus (53·9% with LPS versus 28·5% with hypoxia).