Fig 2: p52 supports viability and spheroid formation in anchorage resistant LA environment. a) Western blot and quantification demonstrating increased p52 protein levels and decreased activated MEK levels in LA relative to HA conditions. b) Loss of p52 or IKKe alone had no effect on viability in anchorage supportive conditions or anchorage resistant conditions after 48 h culture. Loss of both IKKe and p52 significantly reduced survival in the anchorage resistant conditions. Statistical analysis: ** indicates condition significantly different when compared to all HA and LA conditions, one-way ANOVA, post hoc-Tukey. c) Pharmacological inhibition of MEK significantly reduced survival in HA conditions and in the LA conditions when IKKe is also absent. Statistical analysis: * indicates condition significantly different from shNeg with vehicle in LA conditions; # indicates condition significantly different from shNeg with vehicle HA conditions, one-way ANOVA, post hoc-Tukey. d-e) Spheroid formation efficiency at 72 h under LA conditions is supported by p52, but not MEK. Statistical analysis: * indicates condition significantly different from neg shRNA, one-way ANOVA, post hoc-Tukey

Fig 3: IKKe inhibits p52 activity independent of interactions with NIK and IKKa. a) Binding activity of the NF-?B p52 transcription factor was significantly decreased in the presence of IKKe. b) siRNA-mediated knockdown of NFKB2 resulted in a significant decrease of CD44 and CXCL1 mRNA expression. c) siRNA-mediated knockdown of IKBKE resulted in a significant increase in mRNA expression of RELB, NFKB2, and CXCL1. d) CHiP-PCR experiments demonstrate significant enrichment of p52 at the promoter of CXCL1 when IKKe is knocked down. e) Co-immunoprecipitation experiments indicate IKKe does not interact with NIK or IKKa. * significantly different from corresponding shNeg or siNeg control, P < 0.05, two-sided unpaired t-test

Fig 4: No transcriptional NF-?B activity after PrPSc infection. A. Electromobility shift assay revealed NF-?B binding activity in PrPSc-infected neuroblastoma cells (Bos2). Nuclear extracts of Bos2 cells infected with RML6, treated with CD1 or stimulated with 10 ng ml-1 TNF-a were incubated with 32P-labelled double-stranded oligonucleotide specific for NF-?B. The TNF-a sample was incubated with a 50-fold excess of cold NF-?B oligonucleotide as specific competitor or with a mutated oligonucleotide as control. B. ?B precipitation experiment and detection using antibodies directed against p65, p50, p52 and Bcl-3. ?B binding proteins were precipitated from nuclear extracts using an agarose-conjugated consensus ?B oligonucleotide. The precipitates from either untreated controls, TNF-treated or RML6-infected Bos2 cells were divided and analysed in Western blots for the presence of the various NF-?B subunits. The immunoblot was quantified by scanning densitometry. The total signal intensities (p65 + p50, +p52 + Bcl-3) for the treated cells relative to the control cells and the portion of each subunits are given in percentage. C. Luciferase reporter gene assay showed no transcriptional NF-?B activity after PrPSc infection (0.2% or 0.02%), the value of relative light unit (RLU) per µg protein being equivalent for RML6- and CD1-treated cells. Mean values and SEM of three independent transfections are shown.

Fig 5: Reduced Bcl-xL expression correlates with enhanced caspase-3 activation in NF-?B2- and Bcl-3-deficient mice after 11 weeks of PrPSc infection. A. Bcl-xL expression (black arrows), active cleaved caspase-9 and caspase-3 were determined by immunohistological staining. Bcl3–/–, Nfkb2–/–, Nfkb1–/– and C57Bl/6 mice were inoculated with RML6 and three animals per groups were sacrificed at 11 weeks p.i. Brain sections of three animals were analysed for each mouse line. Representative areas of the entorhinal cortex are shown. Magnification ×20. B and C. For quantification of positive cells, three sections of three different mouse brains for each strain were chosen. The graphic represents the amount of positive cells in the brain of the deficient mice compared with the C57Bl/6 mice in percentage of control. SEM was shown as bars. For detailed information on quantification see Experimental procedures.