Figure 6. STAT1, STAT6 and NFκB control of IL4I1 expression.

(A & B) IL4I1 activity in monocytic cells after inhibition of STAT1 expression with fludarabine. THP1 cells were pre-treated or not with 50μM of fludarabine 24h before stimulation with IFNγ. Total STAT1 protein expression (STAT1α and STAT1β) was analyzed by Western blot with untreated THP1 cells as control (A) and IL4I1 activity was measured as in Fig. 3 (B). (C) Role of STAT6 in IL4I1 activity in B cells. LCL cells were transfected with 3 μg control siRNA or STAT6 siRNA 72h prior stimulation with IL4. IL4I1 activity was measured 48h later and reported to control transfected cells left untreated. (D, E & F) Role of NFκ B in IL4I1 activity in monocytic and B cells. LCL cells were transiently transfected with a plasmid vector coding for the dominant IκBα S32A-S36A mutant (IκBα 32/36) or wild type (WT) IκBα as a control. IL4I1 activity was measured at day 3 after the transfection (D). THP1 cell line stably expressing IκBα 32/36 was established by infecting THP1 cells with pTRIP-ΔU3-EF1α-IRES GFP lentiviral vector and sorting of GFP⁺ cells. Iκ Bα 32/36 THP1 cells and control THP1 cells were stimulated by LPS (20 ng/ml for 90 min). Nuclear extracts were prepared and analyzed by EMSA using a ³²P-labeled HIV-LTR tandem κ B oligonucleotide as a probe, to verify that NF-κB DNA binding activity was blocked in IκBα 32/36 THP1 cells (E). IL4I1 activity was measured 48h post-stimulation by LPS Data are presented as percent activity of mutant IκBα-expressing cells in comparison to their respective controls (F). All the IL4I1 activity results are the mean of 3 independent experiments ± SD. * p < 0.05. Note that the cell lysate samples used for all the measures are prepared with a fixed quantity of viable cells to avoid a bias due to variable cell viabilities in the different test conditions.