Journal: The Kurume Medical Journal

Article Title: Effects of Pemafibrate on Reducing Oxidative Stress and Augmenting Angiogenesis in Ischemic Limb Tissue

doi: 10.2739/kurumemedj.ms6934006

Figure Lengend Snippet: Fig. 3. Tissue expression of nuclear factor-erythroid 2-related factor 2 (Nrf2), superoxide dismutase (SOD)-1 and -2, heme oxygenase (HO)-1, and catalase in ischemic lower limbs of control mice and mice adminis- tered pemafibrate. (a) Representative western blotting images of Nrf2, SOD-1, SOD-2, HO-1, catalase, and αactin expression in the ischemic limbs of mice in the control, P-05, and P-10 groups. C = control group; P-05 = P-05 group (mice treated with 0.5 mg/kg/day pemafibrate); and P-10 = P-10 group (mice treated with 1.0 mg/kg/day pemafibrate). Two different samples corresponding to each group were loaded on the same membrane. α-Actin was used as a loading control. (b) Pooled data for Nrf2/α-actin, SOD-1/α-actin, SOD-2/αactin, HO-1/α-actin, and catalase/α-actin expression ratios in the three groups (*p < 0.05 versus the control group; n = 10, each).

Article Snippet: The membranes were blocked with milk buffer (Blocking One; Nacalai Tesque, Inc., Kyoto, Japan) and then incubated overnight at 4 °C with the following primary antibodies: anti-pyruvate dehydrogenase kinase 4 (PDK4) antibody (cat. no. 12949-1-AP; Proteintech, Rosemont, IL, USA), anti-catalase antibody (cat. no. 66765-1-Ig; Proteintech), anti-superoxide dismutase (SOD)-1 and -2 antibodies (cat. nos. sc-101523 and sc-137254, respectively; Santa Cruz Biotechnology, CA, USA), anti-heme oxygenase (HO)-1 antibody (cat. no. 10701- 1-AP; Proteintech), anti-nuclear factor-erythroid 2-related factor 2 (Nrf2) antibody (cat. no. 16396-1-AP; Proteintech), anti-α-actin antibody (cat. no. 23660-1- AP; Proteintech), and anti-glyceraldehyde-3- phosphate dehydrogenase (GAPDH) antibody (cat. no. MAB374; Millipore, Billerica, MA, USA).

Techniques: Expressing, Control, Western Blot, Membrane

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of ALG3 stimulates cancer cell immunogenic ferroptosis to potentiate immunotherapy

doi: 10.1007/s00018-022-04365-4

Figure Lengend Snippet: ALG3 depletion induces ferroptosis. a, b ALG3 knockout significantly deceases the level of reduced glutathione (GSH) in ALG3-KO B16 (a) and 786-O (b) cells. c, d Flow-cytometry estimates ROS levels of subcutaneously grown vector control and ALG3-KO B16 tumors. e Level of malondialdehyde (MDA) in vector control and ALG3-KO B16 tumor tissues. f, i Western blot analysis of the expression of ferroptosis-related proteins in vector control, ALG3-KO (f, g) and Tunicamycin (TM)-treated 786-O cells (h, i). j IHC showed dark brown positive staining for GPX4 and Nrf2 in vector control and ALG3-KO B16 tumors on 11 days after inoculation. Scale bars = 200 µm. k Quantitative analysis of immunohistochemistry staining for j. Data were collected from 5 random fields of each of the 5 tumor samples and present mean ± SEM. p values were determined by unpaired two-sided t test. l Western blot analysis of the expression of ferroptosis-related proteins in ferroptosis inhibitor, ferrostatin-1 (Ferr-1)-treated ALG3-KO B16 cells. m Quantitative analysis of western blots for l. Levels of GSH (n) and ROS (o) in ferrostatin-1 (Ferr-1)-treated ALG3-KO B16 cells. Data in a, b, d, e, g, i, k, m–o present mean ± SEM; n = 2 biological replicates for g, i, m; n = 3 biological replicates for a, b, n, o; n = 4 biological replicates for d, e; p values were determined by unpaired two-sided t test. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Mouse anti-SREBP1 Monoclonal antibody (1:500, Cat No. 66875-1, Proteintech Inc), rabbit anti-Ki67 polyclonal antibody (1:500, Cat No. 27309-1, Proteintech Inc), anti-rabbit Nrf2 monoclonal antibody (1:200, Cat No. 16396-1, Proteintech Inc), or rabbit anti-GPX4 polyclonal antibody (1:200, Cat No. 14432-1, Proteintech Inc).

Techniques: Knock-Out, Flow Cytometry, Plasmid Preparation, Control, Western Blot, Expressing, Staining, Immunohistochemistry

Journal: Advanced Science

Article Title: Nrf2 Drives Epigenetic Reprogramming and Acts as the Master Regulator of KLF4 Expression and Activity in Arsenic‐Induced Transformation

doi: 10.1002/advs.202500221

Figure Lengend Snippet: Nrf2 is essential for As 3+ ‐induced malignant transformation. A) Anchorage‐independent growth of WT and Nrf2 KO cells treated with 0.25 µ m As 3+ for three months in soft agar. Middle panel quantifies cell colonies (>100 µm in diameter) in soft agar from three independent experiments (n = 3 per group per experiment). Right panel shows the proliferation of As 3+ ‐transformed WT and Nrf2 KO cells (n = 6 per group). Data are presented as mean ± SD. Statistical significance was determined using Student's t ‐test (middle panel) or paired one‐way ANOVA with Bonferroni's multiple comparisons test (right panel). B) Representative images of tumors formed by subcutaneous inoculation of As 3+ ‐transformed WT and Nrf2 KO cells in NSG mice (n = 5 per group). The right panel displays quantified ex vivo tumor volumes. Data are presented as mean ± SD. Statistical significance was determined using Student's t ‐test. C) RNA‐seq analysis showing the top 30 most upregulated genes in WT cells after treatment with 2 µ m As 3+ for 12 h. Results are shown as average log2 fold changes (n = 3 per group). D) Enrichr pathway analysis of As 3+ ‐induced genes in WT cells. Results are shown as log p ‐value. E) Profiling of As 3+ ‐induced stemness‐related genes using Stemchecker (top) and cancer pathway analysis using Hallmark Enrichment Plot (bottom). ESC: embryonic stem cell; NSC: neural stem cell; HSC: hematopoietic stem cell; iPSC: induced pluripotent stem cell; MSC: mesenchymal stem cell. F) Identification of Nrf2‐dependent genes by comparing differentially expressed genes between Nrf2 KO and WT cells with or without As 3+ treatment. Bottom panel shows major transcription factor pathways regulating these Nrf2‐dependent genes. G) Reduced expression of stemness‐ and EMT‐ related genes in Nrf2 KO cells. RNA‐seq data were obtained from three independent biological replicates per group. Results are presented as average log2 fold changes (Nrf2 KO versus WT) (n = 3 per group).

Article Snippet: The supernatant was incubated overnight at 4 °C with an Nrf2 antibody (Proteintech, cat#16396‐1‐AP, 1:200), followed by a 2 h incubation with ChIP Grade Protein A/G Magnetic Beads.

Techniques: Transformation Assay, Ex Vivo, RNA Sequencing, Expressing

Journal: Advanced Science

Article Title: Nrf2 Drives Epigenetic Reprogramming and Acts as the Master Regulator of KLF4 Expression and Activity in Arsenic‐Induced Transformation

doi: 10.1002/advs.202500221

Figure Lengend Snippet: Oncogenic properties of Nrf2 in human cancers. A) High levels of Nrf2 predict poorer overall survival in lung cancer patients (left, n = 429) and in cancer patients undergoing anti‐PD1 immune therapy (middle, n = 520). The right panel shows increased expression of the Nrf2 gene in non‐responder group of NSCLC patients subjected to anti‐PD1 immune therapy ( https://www.rocplot.com/immune ). Statistical significance in right panel was determined using Mann‐Whitney test. B) Copy number variations of the Nrf2 gene across different human tumors (left). The right panel shows the association between Nrf2 gene mutation and poorer overall survival in lung cancer patients. C) Molecular Signature Cancer Hallmark pathway analysis of 604 genes positively correlated with Nrf2 (Spearman's Rho ≥ 0.5) in 1865 human lung tumors. (Right) The Top 20 stemness‐associated genes enriched in MYC signaling pathways were among those most highly correlated with Nrf2 expression. D) Nrf2 (NFE2L2) is the top KLF4 ‐correlated genes in human lung cancer and one of the most highly correlated genes with KLF4 in normal human lung tissue. Lower left: Positive correlation of Nrf2 and KLF4 expression across 33 different tumor types in the GEPIA database. Lower right: Positive correlation of Nrf2 and KLF4 expression in 208 lung cancer cell lines from the CCLE. E) High KLF4 expression predicts poor overall survival in lung cancer patients and reduced PFS in patients undergoing anti‐PD1 ICB therapy.

Article Snippet: The supernatant was incubated overnight at 4 °C with an Nrf2 antibody (Proteintech, cat#16396‐1‐AP, 1:200), followed by a 2 h incubation with ChIP Grade Protein A/G Magnetic Beads.

Techniques: Expressing, MANN-WHITNEY, Mutagenesis, Protein-Protein interactions

Journal: Advanced Science

Article Title: Nrf2 Drives Epigenetic Reprogramming and Acts as the Master Regulator of KLF4 Expression and Activity in Arsenic‐Induced Transformation

doi: 10.1002/advs.202500221

Figure Lengend Snippet: KLF4 is a transcriptional target of Nrf2. A) WT and Nrf2 KO cells treated with the indicated concentrations of As 3+ for 6 h followed by Western blotting analysis of the indicated proteins. Quantitative results of KLF4 and MYC are shown below. Data are presented as mean ± SD (n = 3). * p < 0.05 versus control by one‐way ANOVA with Bonferroni's multiple comparisons test. (Right) ARE‐Luciferase Reporter Assay for Nrf2 Activation. ARE‐inducible luciferase reporter assay showing Nrf2 activation over 0–8 h of treatment with 2 µ m As 3+ . Data are presented as mean ± SD (n = 9). B) ChIP‐seq analysis showing As 3+ ‐induced Nrf2 binding and enrichment of enhancer markers H3K4me1 and H3K27Ac at the KLF4 gene locus. ARE elements and their corresponding genomic positions are shown at the top of the panel. Potential enhancers are designated as e1 to e7. The numbers in the bottom right panel indicate the relative degree of enhancer marker enrichment in each group. C) ChIP‐qPCR analysis showing Nrf2 occupancy at the identified ARE elements on the KLF4 locus in transformed WT cells treated with 1 µ m As 3+ for 6 h (WTtr). Data are presented as mean ± SD (n = 9). * p < 0.05 versus WT, determined by Student's t ‐test.

Article Snippet: The supernatant was incubated overnight at 4 °C with an Nrf2 antibody (Proteintech, cat#16396‐1‐AP, 1:200), followed by a 2 h incubation with ChIP Grade Protein A/G Magnetic Beads.

Techniques: Western Blot, Control, Luciferase, Reporter Assay, Activation Assay, ChIP-sequencing, Binding Assay, Marker, ChIP-qPCR, Transformation Assay

Journal: Advanced Science

Article Title: Nrf2 Drives Epigenetic Reprogramming and Acts as the Master Regulator of KLF4 Expression and Activity in Arsenic‐Induced Transformation

doi: 10.1002/advs.202500221

Figure Lengend Snippet: Nrf2 regulates KLF4 expression in As 3+ ‐transformed human iPSCs. A) ChIP‐seq comparison of Nrf2 binding profile at the KLF4 gene locus between BEAS‐2B cells and iPSCs. Shared Nrf2‐binding peaks between the two cell types were highlighted. The lower panel illustrates conserved Nrf2‐binding elements within the KLF4 gene. B) RNA‐seq analysis showing KLF4 induction by As 3+ in iPSCs. Data are presented as mean ± SD (n = 3, based on count values). * p < 0.05 versus WT, determined by Student's t ‐test. C) Chromatin methylation status at the KLF4 gene locus, determined by ChIP‐seq using the indicated antibodies in control and As 3+ ‐transformed iPSCs.

Article Snippet: The supernatant was incubated overnight at 4 °C with an Nrf2 antibody (Proteintech, cat#16396‐1‐AP, 1:200), followed by a 2 h incubation with ChIP Grade Protein A/G Magnetic Beads.

Techniques: Expressing, Transformation Assay, ChIP-sequencing, Comparison, Binding Assay, RNA Sequencing, Methylation, Control

Journal: Advanced Science

Article Title: Nrf2 Drives Epigenetic Reprogramming and Acts as the Master Regulator of KLF4 Expression and Activity in Arsenic‐Induced Transformation

doi: 10.1002/advs.202500221

Figure Lengend Snippet: Elevated potential of KLF4 expression in transformed WT cells. A) Western blotting analysis of Nrf2 activation and expression of KLF4 and MYC in transformed WT and Nrf2 KO cells treated with the indicated concentration of As 3+ . B) Expression levels of genes associated with CSCs in transformed WT cells relative to non‐transformed WT cells. RNA‐seq data were obtained from three independent biological replicates per group. Gene expressions are shown as average log2 fold changes (n = 3 per group). C) Transcription factor pathway assay of upregulated genes in transformed WT cells. D) Semi‐quantitative RT‐PCR showing KLF4 mRNA expression in control and As 3+ ‐treated transformed WT (WTtr) and transformed Nrf2 KO (Nrf2 KOtr) cells (n = 6). Statistical significance was determined by paired Student's t ‐test. E) ChIP‐seq analysis showing histone methylation profiles of the indicated markers at the KLF4 gene locus in non‐transformed (upper panel) and transformed BEAS‐2B cells (bottom panel). Nrf2 ChIP‐seq spectra from control and As 3+ ‐treated non‐transformed BEAS‐2B cells are shown at the top of the panel as positional references for the indicated histone methylation markers. The H3K4me3 peaks at the TSS of KLF4 (highlighted in yellow) and up‐ and down‐ stream H3K4me3 peaks (highlighted in green) are indicated.

Article Snippet: The supernatant was incubated overnight at 4 °C with an Nrf2 antibody (Proteintech, cat#16396‐1‐AP, 1:200), followed by a 2 h incubation with ChIP Grade Protein A/G Magnetic Beads.

Techniques: Expressing, Transformation Assay, Western Blot, Activation Assay, Concentration Assay, RNA Sequencing, Quantitative RT-PCR, Control, ChIP-sequencing, Methylation

Journal: Advanced Science

Article Title: Nrf2 Drives Epigenetic Reprogramming and Acts as the Master Regulator of KLF4 Expression and Activity in Arsenic‐Induced Transformation

doi: 10.1002/advs.202500221

Figure Lengend Snippet: Nrf2 contributes to the establishment of enhancers. A) Schematic diagram illustrating the establishment of poised and active enhancers. The right panel indicates the expression levels of enhancer‐related genes in Nrf2 KO cells relative to WT cells. RNA‐seq data were obtained from three independent biological replicates per group. Expression data are shown as log2 fold changes. B) Western blot analysis showing that knocking out Nrf2 reduces the expression of KMT2D and EP300. C) ChIP‐seq analysis demonstrating that As 3+ enhances the enrichment of Nrf2 and H3K4me3 at TSS of KMT2D in the indicated cells. The conserved Nrf2‐binding ARE element is indicated at the top of the panel.

Article Snippet: The supernatant was incubated overnight at 4 °C with an Nrf2 antibody (Proteintech, cat#16396‐1‐AP, 1:200), followed by a 2 h incubation with ChIP Grade Protein A/G Magnetic Beads.

Techniques: Expressing, RNA Sequencing, Western Blot, ChIP-sequencing, Binding Assay

Journal: Advanced Science

Article Title: Nrf2 Drives Epigenetic Reprogramming and Acts as the Master Regulator of KLF4 Expression and Activity in Arsenic‐Induced Transformation

doi: 10.1002/advs.202500221

Figure Lengend Snippet: Knocking out Nrf2 blocks As 3+ ‐induced genome‐wide KLF4 binding. A) ChIP‐seq heatmaps showing KLF4 binding in control and As 3+ ‐treated WT and Nrf2 KO cells. B) Distribution of KLF4 enrichment on gene loci in the indicated cells. C) Top‐enriched KLF elements in the indicated cells with or without As 3+ treatment. D) Screenshot of KLF4 binding across the entire chromosome 3 in the indicated cells as determined by ChIP‐seq. E) Photomicrographs illustrating the nuclear translocation of KLF4 in WT cells treated with 2 µ m As 3+ for 12 h. Scale bar = 25 µm. F) Western blot analysis of Nrf2, KLF4, and GAPDH in transformed Nrf2 KO (Nrf2 KOtr) cells and Nrf2‐reexpressing (Nrf2oe) Nrf2 KOtr cells following treatment with the indicated concentrations of As 3+ for 6 h. The right panel presents the relative quantification of Nrf2 and KLF4 protein levels in Nrf2 KOtr cells with or without Nrf2 re‐expression. Data are presented as mean ± SD (n = 6 per group). ** p < 0.01 versus Nrf2 KOtr cells transfected with control vector, determined by Student's t ‐test.

Article Snippet: The supernatant was incubated overnight at 4 °C with an Nrf2 antibody (Proteintech, cat#16396‐1‐AP, 1:200), followed by a 2 h incubation with ChIP Grade Protein A/G Magnetic Beads.

Techniques: Genome Wide, Binding Assay, ChIP-sequencing, Control, Translocation Assay, Western Blot, Transformation Assay, Quantitative Proteomics, Expressing, Transfection, Plasmid Preparation

Journal: Advanced Science

Article Title: Nrf2 Drives Epigenetic Reprogramming and Acts as the Master Regulator of KLF4 Expression and Activity in Arsenic‐Induced Transformation

doi: 10.1002/advs.202500221

Figure Lengend Snippet: Identifying KLF4 target genes through ChIP‐seq. A) Volcano plots showing genes regulated by KLF4 in As 3+ ‐treated cells versus control cells (top panel) and in As 3+ ‐treated WT cells versus As 3+ ‐treated Nrf2 KO cells. B) Characteristics of KLF4‐regulated genes in response to As 3+ treatment. C) Top KLF4‐enriched genes induced by As 3+ . Genes marked in red are upregulated in As 3+ ‐transformed cells. Data is shown as average log2 fold changes (n = 3 per group). D) Pathway analysis of KLF4‐regulated genes in response to As 3+ . E) KLF4 binding to Nrf2‐target genes with or without overlapping Nrf2‐binding peaks in Nrf2 ChIP‐seq data.

Article Snippet: The supernatant was incubated overnight at 4 °C with an Nrf2 antibody (Proteintech, cat#16396‐1‐AP, 1:200), followed by a 2 h incubation with ChIP Grade Protein A/G Magnetic Beads.

Techniques: ChIP-sequencing, Control, Transformation Assay, Binding Assay

Journal: Advanced Science

Article Title: Nrf2 Drives Epigenetic Reprogramming and Acts as the Master Regulator of KLF4 Expression and Activity in Arsenic‐Induced Transformation

doi: 10.1002/advs.202500221

Figure Lengend Snippet: Co‐occupancy of Nrf2 and KLF4 facilitates enhancer activation. A) Genome browser tracks showing representative distribution and co‐localization of Nrf2, KLF4, H3K4me1, and H3K27Ac across chromosome 7 (left) and at the EGFR gene locus (right). Green boxes highlight regions of Nrf2 and KLF4 co‐occupy that also exhibit enhancer‐associated histone modifications (H3K4me1 and H3K27Ac), suggesting potential enhancer activity. B) Schematic representation of KLF4 self‐regulation, showing enhanced KLF4 binding within its own gene body and upstream regulatory regions. The red box highlights a region of Nrf2 and KLF4 co‐occupancy overlapping with active enhancer markers. Blue triangles denote predicted KLF4 binding elements, while red filled triangles indicate Nrf2 peaks containing ARE elements. The red unfilled triangle marks an Nrf2 peak lacking a typical ARE element. The green triangle points to the strongest Nrf2 enrichment peak, located downstream of the KLF4 gene in a region devoid of KLF4 binding or H3K27Ac signal.

Article Snippet: The supernatant was incubated overnight at 4 °C with an Nrf2 antibody (Proteintech, cat#16396‐1‐AP, 1:200), followed by a 2 h incubation with ChIP Grade Protein A/G Magnetic Beads.

Techniques: Activation Assay, Activity Assay, Binding Assay