Figure 7: Pellino3 deficiency sensitizes cells to TNF/Smac mimetic-induced apoptosis and TNF/cycloheximide-induced cleavage of RIP1.

(a,b) Immortalized BMDMs from Pellino3^+/+ and Pellino3^−/− mice were treated in the absence (control) or presence of murine TNF (40 ng ml⁻¹) and/or Smac mimetic (10 μM) for 16 h. (c,d) Hela cells were infected with lentivirus containing constructs that encoded control or human Pellino3-specific shRNA. Cells were grown in the presence of puromycin to select for cells showing stable integration of shRNA constructs. Selected cells were treated in the absence (control) or presence of human TNF (40 ng ml⁻¹) and/or Smac mimetic BV6 (10 μM) for 24 h. (a,c) Cells were analysed by flow cytometry for percentage of cells staining positive for annexin V. Data represent the mean +/− s.e.m. of two independent experiments. (b,d) Cell lysates were probed by immunoblotting using antibodies against PARP, cleaved caspase 3, pan-cIAP and β-actin. Cleaved forms of PARP and caspase 3 are indicated by arrows. (e,f) MEFs were isolated from Pellino3^+/+ and Pellino3^−/− embryos and transfected with control or RIP1-specific siRNA. After 48 h, cells were treated in the absence (control) or presence of murine TNF (40 ng ml⁻¹) plus cycloheximide (CHX) (10 μg ml⁻¹) (TC) or murine TNF plus cycloheximide plus ZVAD (20 μM) (TCZ) for 10 h. (e) Cells were analysed by flow cytometry for percentage of cells staining positive for annexin V. Data represent the mean +/− s.e.m. of three independent experiments. **P<0.01; ***P<0.001 (two-way ANOVA). (f) Cell lysates were probed by immunoblotting for expression levels of RIP1, cleaved PARP and β-actin. The autoradiograph for the RIP1 immunblot was exposed for short and long times. Full-length images of immunoblots are shown in Supplementary Fig. S14.