Figure 2
Figure 2. Tax represses WWOX expression through the noncanonical NF-κB pathway. (A) WWOX repression by Tax. Jurkat Tax-TetOn cells were treated with doxycycline (DOX) for the indicated times to induce Tax expression. The induction of Tax protein was analyzed by direct IB (top). The relative levels of WWOX mRNAs in the Tax inducible cells were analyzed by real-time RT-PCR and normalized according to β-actin mRNA level and represented as a percentile of that in untreated cells (arbitrarily set as 100). These data represent mean (± SD) for n = 3 samples. (B) Requirement of p100/p52 in Tax repression of Wwox. Semiquantitative RT-PCR analysis was performed to detect the expression levels of Wwox, TAX, and Gapdh in the matched ear tissues from p100+/+ mice, Tax+p100+/+ mice, p100−/− mice and Tax+p100−/− mice. The relative levels of Wwox mRNAs based on Gapdh mRNAs in Tax+ cells were also represented as a percentile of the levels in Tax−p100+/+ cells (arbitrarily set as 100). (C) Down-regulation of WWOX gene expression by CD40 activation. The CD40+ cell lines KM-H2 and L428 were treated with anti–CD40 antibodies (10μg/mL) followed by real-time RT-PCR analysis as described in panel A. (D) No significant effect on Wwox gene expression by Tax2. MEFs stably expressing Tax2 or an empty vector were subjected to real-time RT-PCR analysis as described in panel A. Tax 2 expression level was examined by IB. (E) RNA repression of WWOX in HTLV-I–transformed T-cell lines and ATL cell lines. The relative levels of WWOX mRNAs in the indicated HTLV-I–transformed T-cell lines and ATL cell lines were analyzed by real-time RT-PCR analysis as described in panel A. (F) Protein repression of WWOX in HTLV-I–transformed T-cell lines and ATL cell lines. Protein expression levels of WWOX were analyzed in the indicated cell lines by IB. The relative levels of WWOX proteins based on Hsp90 proteins were represented as a percentile of levels in Jurkat cells (arbitrarily set as 100). (G) WWOX repression in freshly isolated ATL cells. The levels of WWOX mRNA in PBMCs directly from ATL patients or healthy donors were analyzed by real-time RT-PCR and represented as a percentile of that in normal control No. 1 (arbitrarily set as 100).

Tax represses WWOX expression through the noncanonical NF-κB pathway. (A) WWOX repression by Tax. Jurkat Tax-TetOn cells were treated with doxycycline (DOX) for the indicated times to induce Tax expression. The induction of Tax protein was analyzed by direct IB (top). The relative levels of WWOX mRNAs in the Tax inducible cells were analyzed by real-time RT-PCR and normalized according to β-actin mRNA level and represented as a percentile of that in untreated cells (arbitrarily set as 100). These data represent mean (± SD) for n = 3 samples. (B) Requirement of p100/p52 in Tax repression of Wwox. Semiquantitative RT-PCR analysis was performed to detect the expression levels of Wwox, TAX, and Gapdh in the matched ear tissues from p100+/+ mice, Tax+p100+/+ mice, p100−/− mice and Tax+p100−/− mice. The relative levels of Wwox mRNAs based on Gapdh mRNAs in Tax+ cells were also represented as a percentile of the levels in Tax−p100+/+ cells (arbitrarily set as 100). (C) Down-regulation of WWOX gene expression by CD40 activation. The CD40+ cell lines KM-H2 and L428 were treated with anti–CD40 antibodies (10μg/mL) followed by real-time RT-PCR analysis as described in panel A. (D) No significant effect on Wwox gene expression by Tax2. MEFs stably expressing Tax2 or an empty vector were subjected to real-time RT-PCR analysis as described in panel A. Tax 2 expression level was examined by IB. (E) RNA repression of WWOX in HTLV-I–transformed T-cell lines and ATL cell lines. The relative levels of WWOX mRNAs in the indicated HTLV-I–transformed T-cell lines and ATL cell lines were analyzed by real-time RT-PCR analysis as described in panel A. (F) Protein repression of WWOX in HTLV-I–transformed T-cell lines and ATL cell lines. Protein expression levels of WWOX were analyzed in the indicated cell lines by IB. The relative levels of WWOX proteins based on Hsp90 proteins were represented as a percentile of levels in Jurkat cells (arbitrarily set as 100). (G) WWOX repression in freshly isolated ATL cells. The levels of WWOX mRNA in PBMCs directly from ATL patients or healthy donors were analyzed by real-time RT-PCR and represented as a percentile of that in normal control No. 1 (arbitrarily set as 100).

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