Fig. 2.

Expression of GADD45 family members is regulated by the NF-κB/IκB signaling pathway through c-Myc expression. (A) Real-time PCR analysis of GADD45α, β, and γ after inhibition of NF-κB in different cancer cell lines. Total RNA was collected from DU145, SKBR3, LNCaP, Caki, and UOK cells infected with Ad5CMVβ-gal or Ad5CMVIκB. Each RNA was normalized to GAPDH. (B) Real-time PCR analysis of GADD45α, β, and γ upon activation of NF-κB. Total RNA was collected from U138 MG cells, treated with IL-1β for 6 h, and infected with Ad5CMVβ-gal or Ad5CMVIκB or without Ad infection (control). Each RNA was normalized to GAPDH. (C) Real-time PCR analysis of GADD45α and γ in prostate cancer cells transfected with NF-κB. Total RNA was collected from LNCaP cells transfected with p50 and p65 NF-κB expression vectors or pCI vector as a control. Each RNA was normalized to GAPDH. (D) Western blot analysis of GADD45α, β, and γ after inhibition of NF-κB. Protein extracts were obtained 24, 48, and 72 h after infection with Ad5CMVβ-gal or Ad5CMVIκB. (E) Western blot analysis of c-Myc after inhibition of NF-κB. Protein extracts were obtained 24, 48, and 72 h after infection with Ad5CMVβ-gal or Ad5CMVIκB. (F) Transcriptional activity of the GADD45α promoter. LNCaP cells were transfected with the GADD45α promoter-luciferase construct with the NF-κB p50 and p65 expression vectors or c-Myc expression vector. Luciferase activity was determined 16 h later. Data are means ± SD of two results of one representative transfection normalized to the amount of β-gal expression. Luciferase activity of the GADD45α promoter is shown as percentage of control of the pCI parental vector as indicated on the left. (G) Transcriptional activity of the GADD45α promoter in DU145 cells after infection with Ad5CMVβ-gal or Ad5CMVIκB and transfection of c-Myc expression vector or parental vector. Luciferase activity of the GADD45α promoter is shown as fold induction over the β-gal control.