Figure 6: Pellino3-deficient MEFs show increased apoptosis in response to TNF/cycloheximide co-stimulation.

(a–c) MEFs were isolated from Pellino3^+/+ and Pellino3^−/− embryos and treated in the absence (control) or presence of murine TNF (40 ng ml⁻¹) and cycloheximide (CHX) (10 μg ml⁻¹) for 10 h. (a) Cells were photographed using a phase contrast microscope (bar 20 μm). (b) 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 (two-way ANOVA). (c) Cell lysates were probed by immunoblotting for expression levels of PARP, cleaved caspase 3 and β-actin. (d–f) MEFs, from Pellino3^−/− embryos, were infected with murine stem cell virus (MSCV), lacking (vector) or containing the myc-tagged murine Pellino3 gene (mP3 retrovirus), FHA domain mutant form of mPellino3 (mP3Fm) or RING-like domain mutant form of mPellino3 (mP3Em). Infected cells were treated in the absence (control) or presence of murine TNF (40 ng ml⁻¹) and cycloheximide (CHX) (10 μg ml⁻¹) for 10 h. (d) Cells were photographed using a phase contrast microscope (bar 20 μm) or viewed by fluorescent microscopy to detect infected cells based on expression of MSCV-encoded GFP. (e) Cells were analysed by flow cytometry for percentage of cells staining positive for annexin V staining. Data represent the mean +/− s.e.m. of three independent experiments; ***P<0.001; n.s: not significant (two-way ANOVA). (f) Cell lysates were probed by immunoblotting for expression levels of PARP, cleaved caspase 3, myc-Pellino3, β-actin and GFP. Full-length images of immunoblots are shown in Supplementary Fig. S13.