Fig. 1

Cytotoxic dose TNFα is sufficient to induce apoptosis in a BAD-dependent manner despite concurrent activation of IKK and NF-κB. a, b Various primary wild-type (WT) and Bad^−/− cells were treated without or with non-cytotoxic (5 ng/ml) or cytotoxic (80 ng/ml) dose TNFα for 12 h, as indicated. Apoptotic cells were detected by Annexin V/Propidium iodide (PI) staining and analyzed by flow cytometric analysis (a) or determined by caspase-3 activity assay (b). Hep hepatocyte, Thy thymocyte, Mac macrophage, SPC splenocyte, FB fibroblast. c, d WT and Bad^−/− embryonic fibroblasts were treated without or with various doses of different fractions (F, fraction; F18, fraction number 18 and etc.) of the TNFα preparation (R&D) (TNFα hereinafter for the simplicity unless otherwise specified) for 12 h. Apoptotic cells were determined as described in (a). e WT and Bad^−/− embryonic fibroblasts were treated without or with various doses of TNFα from three different commercial sources (R&D, GeneScript and PeproTech; Supplementary information, Table S4) for 24 h. Apoptotic cells were determined as described in a. 80 ng/ml TNFα from R&D, 500 ng/ml TNFα from GeneScript, 1000 ng/ml TNFα from PeproTech have similar biological activity (Supplementary information, Table S4). f, g Cytotoxic but not non-cytotoxic dose TNFα from the pooled TNFα preparation (f) or cytotoxic dose TNFα from different fractions of the TNFα preparation (g) induced BAD mitochondrial translocation in primary WT hepatocytes (f) and WT fibroblasts (g), as determined by the cytosol [containing cytoskeleton, Cytosol (+C)] and mitochondria fractionation (see “Materials and methods” for details), followed by immunoblotting with corresponding antibodies. β-Actin and COX-2 were used as the marker of the cytosol and mitochondria, respectively. The percentage of IKK-phosphorylated BAD in total cytoplasmic BAD protein was determined (Supplementary information, Figure S9a-S9b), as described in “Materials and methods”. h Cytotoxic and non-cytotoxic dose TNFα induced comparable activation of IKK and NF-κB in primary hepatocytes. Phosphorylation of IKKβ, IκBα and BAD, as well as their protein levels were analyzed by immunoblotting with corresponding antibodies. i Primary WT and Bad^−/− hepatocytes were treated without or with non-cytotoxic or cytotoxic dose TNFα for various durations, as indicated. Total RNA was extracted for quantitative real-time PCR analysis with different primers specifically for cIAP2, IκBα, and IL-6