Fig 2: IKBKE is required for autophagy induced by the ERBB2 breast oncogene. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE, #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 µm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon pharmacological inhibition of IKBKE activity by CYT387 (2 µM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 µm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: IKKe expression is variable and correlates with sensitivity to MEK inhibition in TNBC subtype. a) 30 mg of protein was analyzed in whole cell lysates of breast cancer cells grown to 70% confluence. b-d) 2,000 cells per well were seeded into 96 well plates and allowed to adhere overnight. Inhibitors were added at indicated concentrations and viability assessed via XTT after 72 hours. * IC50 values significantly different between cell lines as indicated, P < 0.05, one-way ANOVA, comparing each cell line individually. Due to the extreme resistance of cell lines MDA MB 453 and BT474, IC50 values could not be reliably calculated, and statistics are therefore not presented

Fig 4: 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 5: IKBKE phosphorylates TPL2 and MEK1 substrates in vitro and MEK1 phosphorylation by MAP3K8 depends on IKBKE. Known phospho-regulatory sites on (A) TPL2 and (B) MEK1. (C) Putative IKK? phosphorylation sites on TPL2 and MEK1 were determined by Phosphonet Kinase Predictor (Kinexus) and these sites were compared to optimal substrate consensus sequences for IKK?. (D) Optimal phosphorylation sites from known IKK? substrates analyzed and some potential phosphorylation sites were determined from Phosphosite algorithm and substrate consensus sequence logo is generated. (E) In vitro kinase assay has been performed with the overexpressed WT or kinase dead (KD) Myc-IKK? pulled out from HEK293 cells, using GST-TPL2367-467 as substrate. (F) Similarly, endogenous TPL2 is IPed from ß-catc.a. vs ß-catc.a.-Ikbke-/- IEC lysates and used for an in vitro kinase assay against his-Mek1KD substrate. (G) Another in vitro kinase assay was performed using si-scramble vs. siIKBKE transfected DLD-1 cells; endogenous TPL2 is immunoprecipitated and used for phosphorylation of his-Mek1KD substrate. IKK?/TPL2 kinase activities in DLD-1 cells were stimulated via 0-1 hour 10 ng/ml LPS stimulation as indicated. (H) Recombinant GST-IKKß and GST-IKK? were used for in vitro kinase assays to compare their kinase activity on GST-TANK, GST-TPL2367-467 and his-MEK1KD substrates.