Viability was more than 98% as assessed by trypan blue exclusion. Rucaparib Peripheral blood mononuclear cells contain 8–12% MN (CD14 reactive) by immunostaining and flourescent activated cell sorter (FACS) analysis. Blood MN were separated from PBMC by negative isolation (Miltenyi, Gladback, Germany). Cells obtained were 80% CD14 reactive. In some experiments, MN were obtained by adherence to plastic. MN thus obtained are 75–90% CD14 reactive. Inhibition of TGF-β signalling by siRNA. First, we assessed the efficacy of transfection of primary human MN by nucleofection. For this purpose, 3 × 106 MN were combined with 1 μg of pmaxGFP
in 100 μl of nucleofection solution and then MN nucleofection was performed per protocol [Amaxa Inc. (http://www.amaxa.com)]. Negative controls included MN in solution that underwent sham nucleofection. Direct microscopy showed that up to 15% of MN were highly flourecent; however, by FACS analysis, Talazoparib supplier up to 80% of MN were successfully nucleofected with pmaxGFP. To inhibit TGF-β signalling, Smad3 siRNA (100 nm) was added to 0.5 × 106 MN culture. In control experiments of gene silencing studies, an unrelated RNA construct was used as control. Smad3 and control siRNA were purchased (Dharmacon, Lafayette, CO, USA). To quantify mRNA expression, real-time RT-PCR (Taqman: Aplied Biosystems, Foster City, CA, USA) using ABI7700 thermocycler was used. Primers and probe for uPAR were
as before , whereas those for uPA, PAI were purchased (ABI Biosystems, Foster City, CA, USA). Quantities of mRNA were determined
using a dilution series of target cDNA in each assay, and expression of target mRNA copies were corrected to the copy numbers of R18 in the same sample. Statistical analysis. Comparisons of multiple measures assessed using cells from the same groups of subjects were evaluated with paired t-tests. P-values of <0.05 were considered significant. To investigate the role of TGF-β signalling in primary human MN, we used siRNA to Smad3 and assessed for genes in the plasmin/plasminogen pathway [uPAR, plasminogen Etofibrate activator inhibitor (PAI) and urokinase plasminogen activator (uPA)] of TGF-β bioactivation. TGF-β mRNA was also assessed. All these genes are induced by TGF-β signalling through Smad3, however, to differing degrees and therefore are likely differently affected by inhibition of TGF-β signalling. A control gene, TNF-α, known not to be under TGF-β control was assessed as control. MN were transfected with siRNA for Smad3 or a control siRNA construct. Four hrs later, recombinant (r) TGF-β (10 ng/ml) was added to wells. Cultures were harvested 24 h later and total RNA harvested and assessed for uPAR, PAI, uPA, TGF-β and TNF-α mRNA. Figure 1 shows a representative (of four) experiments. In four experiments, whereas uPAR expression was induced about 4- to 30-fold by TGF-β, that of PA1 and uPA mRNA were induced very little (1.5–2-fold).