NF-κB Regulates Mesenchymal Transition for the Induction of Non-Small Cell Lung Cancer Initiating Cells
Figure 5
Mesenchymal cells display constitutive NF-κB activity.
Monolayer and 3D cultures of A549 cells were incubated with cytokines for ninety-six hours. (A) Mesenchymal A549 cells display constitutive NF-κB activated pathways, as determined using phospho-specific antibodies to IκBα and RelA. (B) Untreated and TNF and TGFβ stimulated 2D and 3D cultures of A549 cells were harvested and analyzed for expression of NF-κB regulated genes by QRT-PCR. (C and D) Three dimensional cultures of A549.V (vector control) and A549.I (SR-IκB) were incubated for ninety-six hours in the absence or presence of TNF and TGFβ. (C) Immunoblots confirm the expression of the Flag-tagged SR-IκBα in the A549.I line, which successfully blocked nuclear translocation and DNA binding, as measured by EMSA. (D) QRT-PCR confirmed the inability of A549.I cell to upregulate NF-κB-regulated genes following TNF and TGFβ treatment. Immunoblots in Figure 5A are a representative example from three independent experiments. Results in Figure 5B and 5D are calculated mean ± S.D, *p<0.05, N = 3. RNA values were normalized to GAPDH.