Figure 9.

TNF-induced apoptosis of IKKβ(EE)-expressing IEC involves ripoptosome formation and can be inhibited by BHA. (A) WT and Ikkβ(EE)^IEC enteroids were IB analyzed with antibodies to the indicated proteins. (B) Ikkβ(EE)^IEC/A20^ΔIEC enteroids infected with the indicated A20 constructs and preincubated with doxycycline were stimulated without or with TNF (40 μg/mL) for 4 hours and IB analyzed with the indicated antibodies. (C) Ikkβ(EE)^IEC enteroids were incubated with dimethyl sulfoxide, BHA (10 μM), or diphenylene iodonium (5 μM) together with TNF (40 ng/mL) and photographed under brightfield 4 hours later. Original magnification ×200. (D) Lysates of Ikkβ(EE)^IEC enteroids treated with BHA and/or TNF as indicated were IB analyzed after 4 hours. (E) WT and Ikkβ(EE)^IEC enteroids were stimulated without (C) or with TNF together with zVAD (10 μg/mL) (TZ), BHA (TZB), or Nec-1 (50 nM) (TZN) for 2 hours. Complex IIb was isolated by FADD immunoprecipitation and IB analyzed with the indicated antibodies. (F) Ikkβ(EE)^IEC mice were treated with or without BHA (20 mg/kg), challenged with LPS (0.5 mg/kg Escherichia coli O111:B4), and their survival was monitored over a 36-hour period (n = 5 mice per group). (G) Ikkβ(EE)^IEC mice treated as above were analyzed 4 hours after LPS injection. Jejunal sections were stained with either H&E or antibodies to cC-8 or cC-3. Magnification bars: 100 μm. DMSO, dimethyl sulfoxide; DPI, diphenylene iodonium