2). These results suggest that Helios negatively regulates the expressions of these four PKC genes, which JNK inhibitor order are up-regulated by Aiolos. Next, we examined effects of the PMA/ionomycin treatment on viability and DNA fragmentation of Helios−/− and DT40, since PKCs (especially PKC-δ) are involved in the BCR-mediated apoptosis [19] and [34]. Because the expressions of surface IgM (the major component

of BCR) were often altered in DT40 mutants [32], in our studies on the BCR-mediated apoptosis [19], [34] and [35], we have used PMA/ionomycin treatment which bypasses the BCR-proximal signaling and is not influenced by the amounts of surface IgM, as a surrogate of self-antigen. The Helios-deficiency remarkably led to the suppression of the induced apoptosis of DT40 treated with the PMA/ionomycin ( Fig. 3). Notably,

the lack of Aiolos caused drastic decreases in the gene expressions of four PKC (PKC-δ, PKC-ε, PKC-η and PKC-ζ) and accelerated the PMA/ionomycin-induced apoptosis of DT40 cells [19]. Therefore, to know participations of PKCs in the PMA/ionomycin-mediated apoptosis signaling of Helios−/−, we treated Helios−/− with Go6976 (for conventional PKCs) or Rottlerin (for novel and atypical PKCs, mainly PKC-δ) in the presence of PMA/ionomycin. As expected, apoptosis of PMA/ionomycin-treated Helios−/− was significantly accerelated by Rottlerin as compared to Go6976 ( Fig. 4). These results suggest that a certain resistance for the BCR-mediated apoptosis in Helios−/− is preferentially brought via the four PKCs SB431542 (mainly PKC-δ, and probably PKC-ε, PKC-η and PKC-ζ), which were remarkably up-regulated in Helios−/−. In addition, the Helios-deficiency

caused remarkable increase in the O2−-generating activity ( Fig. 5A), although expressions of essential genes of the O2−-generating system (p22-phox, gp91-phox, p47-phox and p67-phox) were unchanged in Helios−/− (data not shown). As expected from findings that PKC-δ was required for full assembly of the O2−-generating system and O2−-generation [39], [40], [41] and [42], the enhanced O2−-generating activity in Helios−/− was remarkably inhibited by Rottlerin ( Fig. 5B). These results revealed that the remarkable effect of the Helios-deficiency on the gene expression of PKC-δ ( Fig. 2) probably resulted in the up-regulation of the O2−-generating activity. Our results in this study reveal that Helios may contribute to the regulation Etoposide molecular weight of the BCR-mediated apoptosis and the O2−-generating activity via transcriptional regulation of PKCs in immature B lymphocytes, probably by inhibiting Aiolos functions. As the Helios-deficiency showed insignificant influences on transcription of Ikaros and Aiolos (Supplementary material Fig. S2), Helios would not regulate gene expressions of other Ikaros family members. Unfortunately, we could not study interactions of Helios protein with other Ikaros family proteins, due to lack of appropriate antibody. The interactions should be elucidated in the future.