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. Author manuscript; available in PMC: 2020 Apr 1.
Published in final edited form as: Cancer Res. 2019 Feb 14;79(7):1353–1368. doi: 10.1158/0008-5472.CAN-18-2383

Figure 4. Inhibition of c-Myc and/or NF-κB and activation of AKT enhance Nrf1 nuclear translocation, restores CC expression, and induces cell death.

Figure 4.

A, Immunoblot analysis of CC in E006AA cells after treatment with DOC (10 nM for 24 hrs) alone or in combination with either c-Myc inhibitor (c-Myc I, 75 µM) or NF-κB inhibitor (NF-κB I, 50 µM) or AKT activator (AKT Act, 5 µM). Actin serves as a loading control. B, Quantification of cell death using Trypan blue assay and caspase-3 activity determination in E006AA cells upon treatment with DOC (for 24 hrs) alone or in combination with either c-Myc inhibitor or NF-κB inhibitor or AKT activator. *p < 0.05 vs DOC treated cells. C, Immunoblot analysis of cleaved PARP (Cl PARP) and cleaved caspase-3 (Cl Casp-3) in E006AA cells treated with DOC alone, c-Myc inhibitor alone, NF-κB inhibitor alone, AKT activator alone or DOC in combination with Myc inhibitor or NF-κB inhibitor or AKT activator. Actin serves as a loading control. D, Immunoblot analysis of Nrf1 in cytosolic and nuclear fractions isolated from E006AA cell treated with DOC (for 24 hrs) alone or in combination with either c-Myc inhibitor or NF-κB inhibitor or AKT activator. LDHB and TBP serve as marker proteins and loading controls for cytosolic and nuclear fractions, respectively. E, Nrf1 binding efficiency with CC promoter in E006AA cells upon treatment with DOC (for 24 hrs) alone or in combination with either c-Myc inhibitor or NF-κB inhibitor or AKT activator using ChIP analysis. LNCaP cells were used as positive controls. F, E006AA cells were transfected with CC promoter constructs (CYCS-Luc or ΔCYCS-Luc) and treated with either c-Myc inhibitor or NF-κB inhibitor or AKT activator, followed by luciferase assay after 24 hrs to detect CC promoter activity. *p < 0.05 vs untreated control, #p< 0.05 vs respective groups. G, c-Myc, p65 subunit of NF-κB and PTEN was knock down in E006AA cells using siRNA. Expression of these proteins and CC was determined using immunoblotting. Actin serves as a loading control. Data represent mean ± SD of 3 independent experiments.