A) Western blot of phospho-p65^S536 in Her2-overexpressing breast cancer cells transfected with siRNA to IKK catalytic subunits. SKBr3 (left), H16N2-Her2 (center) and MDA-MB-453 (right) cells were were transfected with 100 nM siRNA to IKKα and IKKβ and whole cell extracts were prepared after 72 hours and western blot analysis performed. B) NF-κB luciferase reporter assay of SKBr3, H16N2-Her2 and MDA-MB-453 cells transfected with IKK siRNA. Whole cell extracts were prepared 72 hours post-siRNA transfection and luciferase levels were measured. Statistically significant differences were determined by students t-test (*<0.05 **<0.001). Fold change of reporter activity with IKK knockdown is shown relative to scrambled siRNA treated cells. Values are the average of at least 3 experiments. Error bars are ± 1 S.E. Samples are normalized by protein concentration (SKBr3) or renilla (H16N2-Her2 and MDA-MB-453). C) Electrophoretic mobility shift assay (EMSA) of SKBr3 cells transfected with IKK siRNA. Nuclear extracts were prepared after 72 hours. Identities of the bound complexes were determined by super-shift with antibodies to p65 and p50. Non-specific binding complexes are noted with as N.S. D) Kinase assay measuring IKK in vitro phosphorylation of IκBα. SKBr3 cells were transfected with IKK siRNA for 72 hours and IKKγ was immunoprecipitated from 500 μg whole cell extracts. Ability of immunoprecipitated complex to phosphorylate purified GST-IκBα was measured (KA). Amount of IKKα and IKKβ in immunoprecipitated complex (IP) and whole cell extracts (lysate) were measured. Fold change in kinase activity was calculated using pixel densitometry and compared to scrambled siRNA transfected cells.

A) Her2⁺ breast cancer cells were transfected with 100 nM NEMO siRNA and whole cell lysates were collected 72 hours post transfection and western blot analysis of phosphorylated p65 was performed using 25 μg total protein. B) Her2⁺ cell lines were transfected with 100 nM NEMO siRNA and whole cell extracts were prepared 72 hours post-siRNA transfection and luciferase levels were measured. Fold change of reporter activity with IKK knockdown is shown relative to scrambled siRNA treated cells. Values are the average of at least 3 experiments. Error bars are ± 1 S.E. Samples are normalized by protein concentration (SKBr3) or renilla (H16N2-Her2 and MDA-MB-453). C) SKBr3 cells were transfected with 100 nM NEMO siRNA and extracts were isolated after 72 hours and qRT-PCR was performed. Fold change of transcript levels is shown relative to scrambled siRNA treated cells. Error bars are ± 1 S.E. D) Her2-overexpressing breast cancer cells were transfected with 100 nM siRNA to IKKα or IKKβ and whole cell extracts were collected 72 hours post transfection. Levels of p100 and p52 were measured by western blot analysis using 25 μg total protein.

A) Cell proliferation of SKBr3 cells transfected with siRNA to IKKα or IKKβ was measured to for 6 days post-transfection compared to scrambled siRNA treated cells using CellTiter cell viability reagent. Knockdown of IKK by siRNA led to a slight increase in cell proliferation. Error bars represent ± 1 S.D. (B) Cell proliferation of SKBr3 cells treated with PI3K inhibitors LY294002 (10 μM) or EGFR/Her2 inhibitor lapatinib (1 μM) was measured over 3 days. Both inhibitors showed a significant decrease in cell proliferation over a course of 3 days. Error bars represent ± 1 S.D. C) SKBr3 cells were transfected with 100 nM siRNA to IKKα or IKKβ and cell invasion was measured after 48 hours fluorometrically. Statistical significance was measured by student’s T-test (*<0.01, **<0.001). Error bars represent ± 1 S.D.

A) Western blot of phospho-p65^S536 in multiple breast cancer cell lines treated with lapatinib. Breast cancer cell lines were treated with 1 μM dual EGFR/Her2 inhibitor lapatinib or DMSO vehicle control for 12 hours. Western blots were performed with 25 μg protein from whole cell extracts. B) Western blot of phospho-IκBα^S32/36 in SKBr3 cells treated with lapatinib. SKBr3 cells were treated with lapatinib (1 μM) or DMSO control over a course of 24 hours and levels of phospho-IκBα^S32/36 were measured by western blot of 25 μg total protein from whole cell extracts. C) Western blot of phospho-Akt^S473 in SKBr3 cells treated with lapatinib. SKBr3 cells were treated for 12 hours with dual EGFR/Her2 inhibitor lapatinib and levels of phospho-Akt^S473 were measured by western blot of 25 μg protein from whole cell extracts.

(A) Quantitative real-time PCR of multiple genes shows different gene expression profiles upon IKKα or IKKβ knockdown. qRT-PCR was performed on extracts from SKBr3 (black bars) and H16N2-Her2 (gray bars) cells transfected with 100 nM IKKα or IKKβ siRNA for 72 hours. Gene expression levels were normalized to Gus or GAPDH and presented as fold change versus cells transfected with scrambled control siRNA. Values are the average of at least 3 experiments. Error bars are ± 1 S.E. (B) Quantitative real-time PCR of multiple genes upon knockdown of p65 by siRNA. SKBr3 and H16N2-Her2 cells were transfected with 100 nM siRNA for 72 hours and gene expression levels were measured. Fold change of transcript levels is shown relative to scrambled siRNA treated cells. Values are the average of at least 3 experiments. Error bars are ± 1 S.E. C) Quantitative real-time PCR shows inhibition of Her2 by lapatinib blocks NF-κB regulated gene expression. SKBr3 cells were treated with 1 μM lapatinib for 8 or 16 hours and gene expression levels of uPA, IL-6, IL-8, TNF and CCL2 were compared to DMSO treated cells. Fold change of transcript levels is shown relative to scrambled siRNA treated cells. Error bars are ± 1 S.E.

Western blot of phospho-p65 serine 536 from SKBr3 (A), H16N2-Her2 (B) and MDA-MB-453 (C) cells treated with PI3K-inhibitor inhibitor LY294002 for 2 hours. Western blot analysis was performed with 25 μg whole cell extracts. D) Luciferase reporter assay of SKBr3 cells treated with LY294002 overnight. Fold change of reporter activity with PI3K-inhibitor treatment is shown relative to vehicle treated cells. Values are the average of at least 3 experiments. Error bars are ± 1 S.E. Samples are normalized by protein concentration.