BIRC3 is transcriptionally activated by IRF4 and NF-κB and is required for ATL cell growth. (A) ChIP-seq gene tracks representing the binding of IRF4 and p65 as well as the presence of H3K27ac marks near the BIRC3 gene locus in various samples. See the Figure 4D legend for details. (B) The basal protein expression levels of BIRC3, IRF4, and p65, as well as the phosphorylation status of IκBα at Ser 32/36, were analyzed by western blot. β-actin was used as the loading control. (C-D) mRNA expression of BIRC3 on knockdown of IRF4 (C) and p65 (D) was determined by qRT-PCR, using 2 different primer sets (BIRC3 P1 and P2). Expression was normalized to that of the internal control (GAPDH) and is presented as fold-changes compared with the sh-GFP control: as mean of SD of duplicates. *P < .05; **P < .01; ***P < .001 by a 2-sample, 2-tailed t-test compared with the sh-GFP control. (E) BIRC3 protein expression was analyzed by western blotting. Three ATL/HTLV-1-transformed T-cell lines [TL-Om1, MT-2 and ATL-55T(-)] and T-ALL (KOPT-K1) cells were transduced with shRNAs targeting BIRC3 (sh-BIRC3-1 and sh-BIRC3-2), as well as with control GFP shRNA by lentiviral infection. Total protein was harvested 3 days after infection. (F) Cell viability was measured in ATL/HTVL1-transformed T-cell lines [TL-Om1, MT-2, ATL-55T(-)] and T-ALL cell line (KOPT-K1) on days 5 and 7 after transduction with control shRNA (sh-GFP) or shRNAs targeting BIRC3 (sh-BIRC3-1 and sh-BIRC3-2). The relative cell growth rate compared with day 3 (in percentage of sh-GFP) is shown as the mean ± SD of triplicate samples. *P < .05; **P < .01; ***P < .001 by a 2-sample, 2-tailed t-test, compared with the shGFP control.