Journal: Cell Communication and Signaling : CCS
Article Title: B-cell translocation gene 2 mediates crosstalk between PI3K/Akt1 and NF?B pathways which enhances transcription of MnSOD by accelerating I?B? degradation in normal and cancer cells
Figure Lengend Snippet: Activation of NFκB-response element in the 2 nd intron of MnSOD gene by BTG2. (A) MnSOD promoter analysis; Cells (0.5×10 5 /12 wells) were cotransfected with BTG2 cDNA and promoter DNA of MnSOD gene and then subjected to luciferase analysis. Transfection of BTG2 up to 100 ng failed to activate promoter of MnSOD. Sp1 cDNA was employed as a positive control. Lower panel shows protein expressions of the transfected DNAs and loading control. (B) Cells were cotransfected with BTG2 cDNA and the κB-RE before luciferase assay. Expression of MnSOD was significantly increased with transfection of BTG2, indicating the activation of κB-RE by BTG2. Immunoblot analysis shows protein expression of BTG2-HA and loading control. (C) To further investigate the regulation of NFκB activation and MnSOD induction by BTG2 expression, IκBα degradation was examined by immunoblot analysis. Note the degradation of IκBα and MnSOD expression in the BTG2-dependent manner. (D) Transduction of HeLa cells with Ad-BTG2 was performed and then treated with 10 μM of MG132 for 1 h before RT-PCR analysis. MG132 abolished induction of MnSOD expression by BTG2, suggesting the upregulation of MnSOD expression by BTG2 via proteasomal degradation of IκBα. (E) Cell lysates with the LacZ or the BTG2 expressers were subjected to ChIP analysis with anti-p65 antibody, and then the interaction was verified by PCR reaction with the primers written in the Additional file 5 . To verify our analysis, HeLa cells were treated with or without 100 ng of 12- O -tetradecanoylphorbol-13-acetate (TPA) for 2 h and then applied to ChIP assay as a positive and negative control. Unstimulated IgG was employed to exclude the nonspecific interaction. Inputs indicate total amount of κB-RE present in the samples. Note the interactions of p65 with κB-RE only in the BTG2 expressers and the TPA treated positive cells. (F) Immunoblot analyses showing the activation of IKKα/β in the BTG2 overexpressers without any changes in the expression of other anti-oxidant enzymes.
Article Snippet: RT-PCR Total cellular RNAs (1.0 μg) isolated with RNAiso Plus were used for cDNA preparation and then amplified by PCR kit (Takara Inc., Japan); First strand cDNA was synthesized using oligo-dT by reverse transcription reaction in 10 μl of reaction volume.
Techniques: Activation Assay, Luciferase, Transfection, Positive Control, Expressing, Transduction, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Negative Control