Figure 1.

TAK1 deletion induces macrophage cell death with necrotic features (A) WT and TAK1^iKO BMDMs were treated with 0.3 µM 4-OHT or vehicle (ethanol) for 4 days. Expression of TAK1 was analyzed by immunoblotting. (B) WT and TAK1^iKO BMDMs were treated with 0.3 µM 4-OHT or vehicle for indicated days. Cell viability was measured by crystal violet assay. Data are the result of 3 independent experiments and show mean percentages of TAK1^iKO cell viability in each day +/− SD. **p-value < 0.01; two-way ANOVA (C) Cre-alone and TAK1^iKO BMDMs were treated with 0.3 μM 4-OHT for 3 days. Floating and attached cells were collected and stained with annexin V-Pacific Blue and Fixable viability dye eFlour 780, then analyzed on flow cytometer. The results shown are representative of 3 independent experiments. (D) WT and TAK1^iKO BMDMs were treated with 0.3 µM 4-OHT or vehicle (ethanol) together with or without 20 µM zVAD for indicated days. Caspase 3 activation was analyzed by anti-cleaved caspase 3 immunoblotting. Tak1-deficient fibroblasts (TAK1 KO) treated with 20 ng/ml TNF for 6 h was used as positive control for caspase 3 activation. (E) TAK1^iKO BMDMs at 5 days post 4-OHT treatment were analyzed using a transmission electron microscope. Scale bars, 2 μm (top left), 0.2 μm (top right), 1 μm (bottom left) or 0.5 μm (bottom right) as shown. Top images: Arrows indicate swollen mitochondria and enlarged nucleus and cytoplasm. Bottom images: Arrows show disrupted plasma membrane and perforated nuclear envelope and lack of nuclear condensation.