plasmids expressing myc-tagged nrf2 pcdna3-myc3-nrf2  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Nrf2-and p53-inducible REDD2/DDiT4L/Rtp801L confers pancreatic β-cell dysfunction, leading to glucose intolerance in high-fat diet-fed mice

doi: 10.1016/j.jbc.2025.110271

Figure Lengend Snippet: Nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and p53 by oxidative stress in INS-1 cells and pancreatic β-cells in male mice . A , INS-1 cells were incubated with or without 1 mM streptozotocin (STZ) for 3 h. After subcellular fractionation, the cell lysates were analyzed by western blotting using anti-Nrf2, anti-p-p53 (Ser15), anti-p53, anti-triosephosphate isomerase (TPI, cytosol marker), and anti-HistoneH2B (nuclear marker) antibodies. B and C , densitometric analysis of nuclear Nrf2 ( B ) and nuclear p-p53 ( C ). D , immunofluorescent analysis of pancreatic sections from mice treated with STZ (40 mg/kg, four consecutive days). The sections were stained for insulin ( green ), Nrf2 and p53 ( red ), and 4′,6-diamidino-2-phenylindole (DAPI, blue ). Representative images from four independent experiments. Bar = 50 μm. Data are expressed as mean ± SD (n = 3). Asterisks indicate statistically significant differences ( p < 0.05, Student’s t test).

Article Snippet: Plasmids expressing Myc-tagged Nrf2 (pcDNA3-Myc3-Nrf2) ( ) and FLAG-tagged p53 (Flag-p53/pRK5) ( ) were obtained from Addgene (Watertown, MA, USA).

Techniques: Translocation Assay, Incubation, Fractionation, Western Blot, Marker, Staining

Journal: The Journal of Biological Chemistry

Article Title: Nrf2-and p53-inducible REDD2/DDiT4L/Rtp801L confers pancreatic β-cell dysfunction, leading to glucose intolerance in high-fat diet-fed mice

doi: 10.1016/j.jbc.2025.110271

Figure Lengend Snippet: Regulation of the regulated development and DNA damage response 2 ( Redd2 ) expression by nuclear factor erythroid 2-related factor 2 (Nrf2) and p53 in INS-1 β-cells. A and B , INS-1 cells transfected with Nrf2 ( A ) or p53 ( B ) expression vector were incubated for 12 h. Redd2 mRNA expression was determined with quantitative RT-PCR. C–E , INS-1 cells transfected with siNrf2 or sip53 were incubated with or without of 1 mM streptozotocin (STZ) for 6 h. The mRNA expressions of Nrf2 ( C ), p53 ( D ), and Redd2 ( E ) were determined using quantitative RT-PCR. F , Promoter analysis of putative electrophile response elements (EpREs) and putative p53 response elements (p53REs) in mouse Redd2 promoter (−2328/−1) region. G and H , INS-1 cells transfected with Nrf2 or p53 expression vector, a firefly luciferase reporter vector containing the wild-type (WT) or mutant Redd2 promoter (−2328/−1), and the Renilla luciferase reporter vector pGL4.73[ hRluc /SV40] for 24 h. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System. Data are expressed as mean ± SD (n = 4). Asterisks indicate statistically significant differences ( p < 0.05, Student's t test). Different letters indicate statistically significant differences ( p < 0.05, Tukey-Kramer's test).

Article Snippet: Plasmids expressing Myc-tagged Nrf2 (pcDNA3-Myc3-Nrf2) ( ) and FLAG-tagged p53 (Flag-p53/pRK5) ( ) were obtained from Addgene (Watertown, MA, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Incubation, Quantitative RT-PCR, Luciferase, Mutagenesis, Reporter Assay

Journal: The Journal of Biological Chemistry

Article Title: Nrf2-and p53-inducible REDD2/DDiT4L/Rtp801L confers pancreatic β-cell dysfunction, leading to glucose intolerance in high-fat diet-fed mice

doi: 10.1016/j.jbc.2025.110271

Figure Lengend Snippet: Interaction of nuclear factor erythroid 2-related factor 2 (Nrf2) and p53 on the promoter of the regulated development and DNA damage response 2 ( Redd2 ) in INS-1 β-cells . A , INS-1 cells were transfected with Nrf2 and/or p53 expression vector by electroporation for 24 h and Redd2 expressions were determined. B–G , INS-1 cells were transfected with Nrf2 and/or p53 expression vector, a firefly luciferase reporter vector of wild type or mutant mouse Redd2 promoter (−2328/−1) (WT ( B ), EpRE2 mutant ( C ), p53RE1 mutant ( D ), or EpRE2/p53RE1 mutant ( E )), EpRE reporter vector (F), or 2xp53RE reporter (G) and the Renilla luciferase reporter vector (pGL4.74[ hRluc /TK] or pGL4.73[ hRluc /SV40]) for 24 h. Luciferase activities were determined using Dual-Luciferase reporter assay system. H – K , ChIP assay was performed using control IgG, anti-p-Nrf2 IgG, and anti-p-p53 IgG. The co-precipitated DNA was subjected to PCR using primers that amplify Redd2 promoter containing the EpRE2 ( H ) or p53RE1 ( J ) elements in rat INS-1 cells. Densitometry of relative amplified DNA bands containing the EpRE2 ( I ) and p53RE1 ( K ). L , GST pull-down assay with purified GST or GST-Myc3-Nrf2 and His-Flag-p53. Pulled down samples were analyzed with western blotting with anti-GST and anti-Flag antibodies. Data are expressed as mean ± SD (n = 4). Asterisk indicates statistically significant differences ( p < 0.05, Dunnett’s test). Different letters indicate statistically significant differences ( p < 0.05, Tukey-Kramer's test).

Article Snippet: Plasmids expressing Myc-tagged Nrf2 (pcDNA3-Myc3-Nrf2) ( ) and FLAG-tagged p53 (Flag-p53/pRK5) ( ) were obtained from Addgene (Watertown, MA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, Electroporation, Luciferase, Mutagenesis, Reporter Assay, Control, Amplification, Pull Down Assay, Purification, Western Blot