Fig. 5.

DNA double-strand breaks stimulate TAK1 kinase activity, and TAK1 inhibition impairs the NF-κB response and sensitizes cells to genotoxic stimuli. (A) Multiple DNA-damaging agents stimulate TAK1 kinase activity. Human U2OS tumor cells were left untreated or treated with TNF-α (10 ng/ml) for 10 min, doxorubicin (Dox; 10 μg/ml), or etoposide (Etop; 40 μM) or exposed to 10 Gy gamma irradiation (IR) for the time periods indicated. TAK1 activity was measured by immunoblotting with a (IB) phospho-specific TAK1 antibody. Total TAK1 levels were measured by immunoblotting with a TAK1 antibody. (B) The NF-κB response to genotoxic stress is impaired in Tak1-deficient MEFs. SV40T-immortalized wild-type (Wt) or Tak1-deficient murine embryonic fibroblasts were left untreated, treated with TNF-α, or treated with doxorubicin for the time periods indicated, and NF-κB activity was measured by monitoring IκBα degradation. Cell lysates were probed with anti-β-actin antibody to ensure that equal amounts of total protein were loaded. IκBα degradation was quantified using densitometry, and values are shown for each lane. (C) Tak1-deficient MEFs are hypersensitive to doxorubicin treatment. Wild-type or Tak1-deficient MEFs were treated with doxorubicin for 24 and 48 h, and viable cells were quantified by trypan blue exclusion. Samples were assayed in triplicate; error bars represent the standard deviations. The experiment whose results are shown is representative of at least three. Statistical evaluation was performed using the unpaired Student t test. **, P < 0.01. (D) Treatment with a TAK1 kinase inhibitor impairs the NF-κB response to DNA damage. Human U2OS cells were pretreated with the TAK1 kinase inhibitor 5Z7 (Bioaustralis) (1 μM) or dimethyl sulfoxide (DMSO) for 2 h. The cells were left untreated or treated with TNF or doxorubicin for the time periods indicated. NF-κB activation was measured by immunoblotting the cell lysates with an IκBα antibody. Total protein levels were determined by immunoblotting with an anti-β-actin antibody. (E) TAK1 kinase inhibition sensitizes human tumor cells to doxorubicin treatment. Human MCF7 tumor cells were pretreated with the TAK1 kinase inhibitor 5Z7 (1 μM) or DMSO control for 2 h. Cells were then left untreated or treated with doxorubicin for 48 h. Viable cells were quantified by trypan blue exclusion. Samples were assayed in triplicate; error bars represent the standard deviations. The experiment whose results are shown is representative of at least three. Statistical evaluation was performed using the unpaired Student t test. *, P < 0.05.