2B), suggesting that also this cytokine gene is directly or indirectly targeted by NAB2. To validate our findings that exogenous NAB2E51K diminishes TRAIL induction, we transfected CAL-1 cells with siRNA against NAB2. We achieved a mere 30%

reduction in NAB2 expression with siRNA, possibly due to the high basal expression levels of NAB2 (Supporting Information Fig. 3B). Nonetheless, we observed a slight reduction of CpG-mediated TRAIL induction, while the induction of CD40 LBH589 concentration remained unaffected (Supporting Information Fig. 3A and C). We next determined whether the reduced TRAIL expression in CAL-l-1-NAB2E51K cells affected their capacity to induce cell death. Compared with CAL-l-1-NAB2 or CAL-l-1-EV, CpG-activated CAL-l-1-NAB2E51K cells were indeed less potent in inducing apoptosis of TRAIL-sensitive Jurkat cells as assessed by AnnexinV expression of the target cells (Fig. 3F; p = 0.020 and p = 0.009). Similar results were found when Caspase-3 activation was measured in Jurkat cells (Supporting Information Fig. 4). Together, these results demonstrate that NAB2 is directly involved in TRAIL induction upon TLR9/7-mediated Seliciclib clinical trial pDC activation, and that blocking

its activity diminishes pDC cytotoxicity. We next assessed which molecules mediate NAB2-dependent TRAIL induction in pDCs. Therefore, we blocked PI3K, p38MAPK, or NF-κB signaling in CpG-activated CAL-1 cells with inhibitors chosen based on their activity without compromising the cell viability (Supporting Information Fig. 5A and B). Interestingly, PI3K signaling was essential for NAB2

induction upon CpG stimulation of pDCs as determined by pretreating CAL-1 cells with the inhibitor PI-103 (Fig. 4A; p < 0.0001). PI-103-treated CAL-1 cells also failed to express TRAIL (Fig. 4C and E), supporting our hypothesis that NAB2 induces TRAIL expression in pDCs. Importantly, the induction of NAB2 and TRAIL mRNA was also significantly blocked by PI-103 in primary pDCs upon CpG stimulation (Fig. 4B and D; p < 0.01 and p < 0.05). Of note, the PI3K-mediated NAB2 induction was independent of mTOR as treatment with Rapamycin did not significantly block the increase of NAB2 mRNA upon CpG stimulation (Supporting Cyclin-dependent kinase 3 Information Fig. 5C). Blocking p38MAPK with SB203580, or blocking NF-κB with BAY11-7082 had no effect on NAB2 induction (Fig. 4A; p = 0.38 and p = 0.09). However, p38MAPK inhibition significantly blocked TRAIL expression (Fig. 4C and E; p < 0.01), suggesting that (i) p38MAPK acts independently of PI3K/NAB2 signaling to induce TRAIL, or that (ii) p38MAPK feeds into the same signaling pathway, but downstream of NAB2 activity. In conclusion, we show that PI3K signaling is required for CpG-mediated NAB2 expression and its downstream target TRAIL. We observed that NAB2E51K only partially blocked TRAIL induction. Interestingly, CAL-1-NAB2E51K cells displayed two peaks of TRAIL expression rather than a uniform decrease (Fig. 3C).