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

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Left, Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with control vector or Myc-SDCBP-expressing vector in the presence of CHX protein synthesis inhibitor at various time points. Right, quantification of BACH1 protein levels using densitometry ( n = 3). ( B ) Free heme level in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector ( n = 3). ( C ) Free heme level in scramble and in SDCBP - KO MDA-MB-231 cells ( n = 3). ( D ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with control vector or NRF2 (encoded by NFE2L2 )-expressing vector. HO-1 protein expression was considered as the positive control. ( E ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with a control or a HO-1 (encoded by HMOX1 )-expressing vector. ( F ) Western blot showing BACH1 protein expression in Hs578T cells transfected with scramble or HO-1 siRNA. ( G ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with the HO-1 or the catalytic inactive HO-1 mutant (H25A) plasmid. ( H ) Western blot showing BACH1 and SDCBP protein expression in MDA-MB-231 cells transfected with scramble or HOIL1 siRNA. ( I ) Western blot showing endogenous FBXO22 protein expression in several breast cancer cells. ( J ) Immunoprecipitation showing the interaction of BACH1 with FBXO22 in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ns: none specific. ( K ) In vivo ubiquitylation assay showing the increase in the polyubiquitylation of BACH1 by FBXO22 overexpression in HEK293 cells transfected with the indicated plasmids. ( L ) In vivo ubiquitylation assay showing the increase in the polyubiquitylation of BACH1 by FBXO22 overexpression in MDA-MB-231 cells transfected with the indicated plasmids. ( M ) In vivo ubiquitylation assay showing the decrease in FBXO22-mediated polyubiquitylation of BACH1 by SDCBP overexpression in HEK293 cells transfected with the indicated plasmids. Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( A – C ). All experiments were repeated at least three times unless otherwise indicated. P values less than 0.05 were considered statistically significant.

Article Snippet: pcDNA3.1 FLAG NRF2 , Addgene , Cat#36971.

Techniques: Western Blot, Expressing, Transfection, Control, Plasmid Preparation, Positive Control, Mutagenesis, Immunoprecipitation, In Vivo, Ubiquitin Assay, Over Expression, Two Tailed Test

Journal: bioRxiv

Article Title: Pyrimethamine and a potent analogue WCDD115 inhibit NRF2 by suppressing DHFR and one-carbon metabolism

doi: 10.1101/2025.02.13.637433

Figure Lengend Snippet:

Article Snippet: To generate doxycycline-inducible NRF2 shRNA KYSE70 cells, we obtained a dox-inducible short hairpin RNAs (shRNAs) targeting human NRF2 from Addgene: #136584, with a sequence of AGA-GCA-AGA-TTT-AGA-TCA-TTT- CTG-CAG-AAA-TGA-TCT-AAA-TCT-TGC-TCT.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Pyrimethamine and a potent analogue WCDD115 inhibit NRF2 by suppressing DHFR and one-carbon metabolism

doi: 10.1101/2025.02.13.637433

Figure Lengend Snippet: A. H1299 NQO1-eYFP cells were treated with DMSO vehicle or 10 µM PYR in the presence of PRL295, CDDOme, or sulforaphane. B. Western blot analysis showing dose-dependent effects of PYR on indicated proteins in KYSE70 cells. C. Western blot analysis of PYR (10 µM) time course effects on indicated proteins in KYSE70 cells. Quantitation across biological triplicate experiments is shown below, normalized to VCL (vinculin) (*P < 0.05 by one-way ANOVA). D. Western blot analysis of lung and esophageal cancer cells following 48h treatment with PYR (10µM). Quantitation across biological triplicate experiments is shown below, normalized to VCL (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA). E-F. Western blots assessing the effects of PYR (10µM; 48h) on the indicated proteins in KYSE70, KYSE450, or KEAP1 KO derivatives. Quantitation across biological triplicate experiments is shown below (****P < 0.0001 by one-way ANOVA). G. Graph showing NRF2 mRNA fold change in KYSE70 cells following 24 and 48h treatment with PYR (10µM). NRF2 expression was normalized to RPL13 A. Data are presented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001) by one-way ANOVA (n ≥ 3 biological replicates per group). H. KYSE70 cells harboring a dox-inducible NRF2 shRNA were treated with the indicated amount of dox for 24h before qPCR and western blot analysis for NRF2. NRF2 protein was normalized to VCL; NRF2 mRNA expression was normalized to RPL13A. Data are presented as means ± SD (*P < 0.05, **P < 0.01, ***P < 0.001) by one-way ANOVA (n ≥ 3 biological replicates per group). See also .

Article Snippet: To generate doxycycline-inducible NRF2 shRNA KYSE70 cells, we obtained a dox-inducible short hairpin RNAs (shRNAs) targeting human NRF2 from Addgene: #136584, with a sequence of AGA-GCA-AGA-TTT-AGA-TCA-TTT- CTG-CAG-AAA-TGA-TCT-AAA-TCT-TGC-TCT.

Techniques: Western Blot, Quantitation Assay, Expressing, shRNA

Journal: bioRxiv

Article Title: Pyrimethamine and a potent analogue WCDD115 inhibit NRF2 by suppressing DHFR and one-carbon metabolism

doi: 10.1101/2025.02.13.637433

Figure Lengend Snippet: A-C. H1299 NQO1-eYFP cells were treated with increasing doses of PYR or WCDD104 in the presence of PRL295, CDDOme or 2 µM SULF. eYFP intensity was normalized to cell number (mCherry positive) before normalization to DMSO vehicle. D. Quantified data from Western blots shown in (**P < 0.05, ***P < 0.01, ****P < 0.001,) by one-way ANOVA (n=4 biological replicates per group). E-F. Western blot analysis of KYSE70 and KYSE450 cells and their KEAP1 KO derivatives after 48h treatment with 10µM PYR, 1µM 115, or 0.1µM MTX. Below, quantitative, normalized data are plotted for NRF2/VCL.

Article Snippet: To generate doxycycline-inducible NRF2 shRNA KYSE70 cells, we obtained a dox-inducible short hairpin RNAs (shRNAs) targeting human NRF2 from Addgene: #136584, with a sequence of AGA-GCA-AGA-TTT-AGA-TCA-TTT- CTG-CAG-AAA-TGA-TCT-AAA-TCT-TGC-TCT.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Pyrimethamine and a potent analogue WCDD115 inhibit NRF2 by suppressing DHFR and one-carbon metabolism

doi: 10.1101/2025.02.13.637433

Figure Lengend Snippet: A-C. H1299 NQO1-eYFP cells were treated with increasing doses of the indicated DHFR inhibitors in the presence of PRL295, CDDOme, or SULF. eYFP intensity was normalized to cell number (mCherry positive) and DMSO vehicle. D, E. Western blot analysis of KYSE70 and A549 cells treated with PYR (10µM), WCDD115 (1µM), MTX (0.1µM), PEM (100nM), or CG (10μM) for 48h. Quantitation and statistical analysis is shown in . F. NRF2 mRNA was quantified by qPCR after 48h treatment of KYSE70 cells with PYR (10µM), WCDD115 (1µM), MTX (0.1µM). Data are normalized to RPL13A and plotted as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001) by one-way ANOVA. G. Dose-response relationships for PYR, WCDD115, and MTX on the enzymatic activity of recombinant hDHFR (3E-3 units). Data are normalized to vehicle and presented as mean ± SD (n=7, ****P< 0.001) by one-way ANOVA. H. Targeted metabolomic profiles of KYSE70 cells treated with PYR (10µM), WCDD115 (1µM), MTX (0.1µM) for 48h. Data shown are fold changes from DMSO (see also Table S3). I. Glutamine, Serine, and NADH levels following 48h treatment of KYSE70 cells with PYR (10µM), WCDD115 (1µM), and MTX (0.1µM). Data are presented as means ± SD (*P < 0.05, **P < 0.01, ***P < 0.001) by one-way ANOVA. J. H1299 and KYSE70 cells were treated with PYR (10µM), WCDD115 (1µM), and MTX (0.1µM) 48 h ± 20µM menadione for the last 3h. ROS was quantified using CellRox. Data are presented as means ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P< 0.001) by one-way ANOVA (n≥3 biological replicates per group).

Article Snippet: To generate doxycycline-inducible NRF2 shRNA KYSE70 cells, we obtained a dox-inducible short hairpin RNAs (shRNAs) targeting human NRF2 from Addgene: #136584, with a sequence of AGA-GCA-AGA-TTT-AGA-TCA-TTT- CTG-CAG-AAA-TGA-TCT-AAA-TCT-TGC-TCT.

Techniques: Western Blot, Quantitation Assay, Activity Assay, Recombinant

Journal: bioRxiv

Article Title: Pyrimethamine and a potent analogue WCDD115 inhibit NRF2 by suppressing DHFR and one-carbon metabolism

doi: 10.1101/2025.02.13.637433

Figure Lengend Snippet: A. Western blot analysis of KYSE70 cells treated with PYR (10 µM), WCDD115 (1 µM), MTX (0.1 µM), PEM (0.1 µM), or CG (10 µM) in the absence or presence of folinic acid (10mg/ml). Data are presented as means ± SD (*P < 0.05, **P < 0.01) by one-way ANOVA (n=3 biological replicates per group). B. KYSE70 parental cells or two monoclonal DHFR KO derivatives were treated with WCDD115, HT, or both for 48 h. Protein expression of DHFR, NRF2, and downstream targets SLC7A11, HMOX1, NQO1, GCLC, and VINC was quantified by Western blot. Quantitative data of biological triplicate experiments are plotted below as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001) by one-way ANOVA. C. KYSE70 cells were treated with the indicated combinations of WCDD115, hypoxanthine (H), and thymidine (T) for 48h. Protein expression of DHFR, NRF2, and downstream targets SLC7A11, HMOX1, NQO1, GCLC, and VINC was quantified by Western blot. Quantitative data of biological triplicate experiments are plotted below as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001) by one-way ANOVA.

Article Snippet: To generate doxycycline-inducible NRF2 shRNA KYSE70 cells, we obtained a dox-inducible short hairpin RNAs (shRNAs) targeting human NRF2 from Addgene: #136584, with a sequence of AGA-GCA-AGA-TTT-AGA-TCA-TTT- CTG-CAG-AAA-TGA-TCT-AAA-TCT-TGC-TCT.

Techniques: Western Blot, Expressing