5 mM CaCl2, 5 mM glucose and 0.03% BSA were stimulated with 200 ng/ml PMA at 37 °C, and the O2− generation was measured by a TD-20/20 luminometer (Promega). To generate Helios-deficient DT40 mutants,

Helios−/−, we first transfected DT40 cells with the targeting construct carrying hygromycin resistance gene ( Fig. 1A). After integration of this targeting vector into one Helios allele, the stable positive transfectants were selected based on both the resistance to hygromycin and the generation of the hybridized 7.3-kb HindIII fragment with probe Helios, in addition to the endogenous 4.0-kb HindIII fragment ( Fig. 1B). One of these clones (−/+) obtained was chosen for second round of transfection with the targeting construct carrying blasticidin S resistance gene. As expected, in the two analyzed clones (clones-1 and 2 of Helios−/−), the probe Helios newly hybridized to the 5.3-kb HindIII fragment, in addition to the 7.3-kb HindIII Selleckchem PFI-2 fragment, with disappearance of the endogenous 4.0-kb HindIII fragment ( Fig. 1B). Similar results were obtained with many other Helios−/− clones (data not shown). Helios isoforms were not expected to be detected in resulting gene-targeted clones, since they lack exon 8 of the chicken Helios gene (corresponding to INK 128 price exon 7 of the human Helios gene [9]), encoding the C-terminal fingers mediating

dimerization, which are contained in all Helios isoforms. To determine whether or not Helios was really disrupted in these DT40 mutants, we measured the steady-state level of Helios mRNA by semiquantitative RT-PCR using 3-oxoacyl-(acyl-carrier-protein) reductase primers corresponding to the exon 8. As expected, no band was detected for the homozygous mutants, Helios−/− tested ( Fig. 1C). The growth rate of Helios−/− clones was not changed (data not shown). Ikaros family members including Helios can dimerize with themselves and/or other members, and are transcribed as several isoforms based on alternative splicing [9]. As the expression level of Helios was very low in DT40 (see many PCR cycle numbers in Fig. 1C), Helios was thought to function as heterodimer

(rather than as homodimer) with other family members. We reported that the Aiolos-deficiency caused changes in the expressions of several genes: bak, caspase-9, ICAD and PKCs (PKC-α, PKC-β, PKC-δ, PKC-ε, PKC-η and PKC-ζ) [19]. To know influences of the Helios-deficiency on these gene expressions, we first carried out semiquantitative RT-PCR on total RNAs prepared from Helios−/−, Aiolos−/− and DT40. We analyzed two independent Helios−/− clones. The Helios-deficiency showed significant influences on transcriptions of several PKCs: PKC-δ (to ∼330%), PKC-ε (to ∼190%), PKC-η (to ∼380%) and PKC-ζ (to ∼360%) except for PKC-α and PKC-β ( Fig. 2, Supplementary material Fig. S1), but insignificant influences on those of bak, caspase-9 and ICAD (data not shown). On the other hand, the Helios-deficiency showed insignificant influences on transcription of Ikaros and Aiolos (Supplementary material Fig. S2).