Journal: bioRxiv

Article Title: Fibroblast Nrf2 inhibits profibrotic transcription with Ddx54 and mitigates pathological fibrosis in the mouse heart and kidney

doi: 10.1101/2023.11.09.566496

Figure Lengend Snippet: A , Western blot to confirm knockout of Keap1 and/or Nrf2 in C2C12 cells. B , Quantitative PCR analysis of representative fibrosis-related genes in Keap1 and/or Nrf2 knockout C2C12 cells 24 hours after TGF-β treatment. C , Western blot to confirm the knockdown of Keap1 and/or Nrf2 in mouse embryonic fibroblasts (MEFs) using siRNA. D , Quantitative PCR analysis of Ctgf and Postn mRNA levels in Keap1 and/or Nrf2 knockdown MEFs 24 hours after TGF-β treatment. E , Quantitative PCR analysis of Postn and Acta2 mRNA levels in Keap1 or Ctgf knockout C2C12 cells at 24 hours after TGF-β treatment. F , Flow cytometry for CellRox to evaluate ROS production in Keap1 knockout C2C12 cells under 24-hour stimulation with TGF-β. N-acetylcysteine (NAC) treatment for 4 hours was employed as an antioxidant control. G , Quantitative PCR analysis of Ctgf, Postn , and Fn1 mRNA levels in Keap1 knockout C2C12 cells stimulated with TGF-β for 24 hours or NAC for 4 hours. Data are presented as mean±SD. P values were calculated by one-way ANOVA with Tukey’s multiple comparison test (n=3).

Article Snippet: Membranes were then probed with anti-Nrf2 (CST, 12721S, 1:1000 for WB), anti-Keap1 (MBL, M-224-3, 1:1000 for WB), anti-Smad3 (CST, 9523S, 1:1000 for WB), anti-Avidin HRP (Biolegend, 405103, 1:2500 for WB), anti-HA tag (CST, 2367, 1:1000 for WB) and anti-β-actin (Sigma, 2228, 1:2000 for WB) antibodies followed by incubation with HRP-conjugated secondary antibodies (CST, 7076S/7074S, 1:1000 for WB).

Techniques: Western Blot, Knock-Out, Real-time Polymerase Chain Reaction, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: Fibroblast Nrf2 inhibits profibrotic transcription with Ddx54 and mitigates pathological fibrosis in the mouse heart and kidney

doi: 10.1101/2023.11.09.566496

Figure Lengend Snippet: A , Representative chromatin landscapes around Ctgf , Fn1 and Hmox1 regions of the mouse genome generated by CUT&Tag using anti-Nrf2 antibody in Keap1 knockout C2C12 cells and Keap1 knockout RAW264.7 macrophages with or without TGBF-β treatment. B , The result of GO annotation analysis. Nrf2-bound gene loci associated with fibrosis-related gene sets, such as extracellular matrix in C2C12 cells. C , Representative chromatin landscapes around Ctgf and Fn1 regions of the mouse genome generated by CUT&Tag using anti-Smad3 antibody in C2C12 cells and Keap1 knockout C2C12 cells with TBF-β treatment. D , Smad3 ChIP-qPCR analysis of C2C12 cells or Keap1 knockout C2C12 cells with or without TGB-β treatment. Primers were designed from the peak and intron regions of Smad3 binding. E , RNA polymerase II (Pol II) ChIP-qPCR analyses of C2C12 cells or Keap1 knockout C2C12 cells with or without TGB-β treatment. Primers were created from the peak and intron regions of Smad3 binding. Data are presented as mean±SD. P values were calculated by two-way ANOVA with Tukey’s multiple comparison test (n=3).

Article Snippet: Membranes were then probed with anti-Nrf2 (CST, 12721S, 1:1000 for WB), anti-Keap1 (MBL, M-224-3, 1:1000 for WB), anti-Smad3 (CST, 9523S, 1:1000 for WB), anti-Avidin HRP (Biolegend, 405103, 1:2500 for WB), anti-HA tag (CST, 2367, 1:1000 for WB) and anti-β-actin (Sigma, 2228, 1:2000 for WB) antibodies followed by incubation with HRP-conjugated secondary antibodies (CST, 7076S/7074S, 1:1000 for WB).

Techniques: Generated, Knock-Out, Binding Assay, Comparison

Journal: bioRxiv

Article Title: Fibroblast Nrf2 inhibits profibrotic transcription with Ddx54 and mitigates pathological fibrosis in the mouse heart and kidney

doi: 10.1101/2023.11.09.566496

Figure Lengend Snippet: A , Western blot to confirm input and immunoprecipitated samples from Nrf2-HA-TurboID cells and (Tet-On) HA-TurboID-NLS cells used for BioID analysis. B , The result of BioID. 91 high-confidence Nrf2 interactors were identified. C , Quantitative PCR analysis of Ctgf , Postn , and Acta2 mRNA levels in Keap1 and/or Ddx54 knockout C2C12 cells 24 hours after TGF-β treatment. Data are presented as mean±SD. P values were calculated by one-way ANOVA with Tukey’s multiple comparison test (n=4).

Article Snippet: Membranes were then probed with anti-Nrf2 (CST, 12721S, 1:1000 for WB), anti-Keap1 (MBL, M-224-3, 1:1000 for WB), anti-Smad3 (CST, 9523S, 1:1000 for WB), anti-Avidin HRP (Biolegend, 405103, 1:2500 for WB), anti-HA tag (CST, 2367, 1:1000 for WB) and anti-β-actin (Sigma, 2228, 1:2000 for WB) antibodies followed by incubation with HRP-conjugated secondary antibodies (CST, 7076S/7074S, 1:1000 for WB).

Techniques: Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction, Knock-Out, Comparison

Journal: bioRxiv

Article Title: Fibroblast Nrf2 inhibits profibrotic transcription with Ddx54 and mitigates pathological fibrosis in the mouse heart and kidney

doi: 10.1101/2023.11.09.566496

Figure Lengend Snippet: A , Generation of a new allele of Keap1 in which exon 3 are flanked by loxP sites. Cre-mediated recombination of the loxP sites resulted in deletion of exon 3. B , PCR evaluation of genotyping for exon 3 deletion in candidate mice. C , Representative Pulsed Wave Doppler images from echocardiography 4 weeks after sham or TAC surgery.

Article Snippet: Membranes were then probed with anti-Nrf2 (CST, 12721S, 1:1000 for WB), anti-Keap1 (MBL, M-224-3, 1:1000 for WB), anti-Smad3 (CST, 9523S, 1:1000 for WB), anti-Avidin HRP (Biolegend, 405103, 1:2500 for WB), anti-HA tag (CST, 2367, 1:1000 for WB) and anti-β-actin (Sigma, 2228, 1:2000 for WB) antibodies followed by incubation with HRP-conjugated secondary antibodies (CST, 7076S/7074S, 1:1000 for WB).

Techniques:

Journal: bioRxiv

Article Title: Fibroblast Nrf2 inhibits profibrotic transcription with Ddx54 and mitigates pathological fibrosis in the mouse heart and kidney

doi: 10.1101/2023.11.09.566496

Figure Lengend Snippet: A , Keap 1 fl/fl mice were crossed with Postn-MerCreMer ( Postn MCM ) mice to generate tamoxifen-inducible fibroblast-specific Keap1 knockout mice. Adult 9-week-old male mice were subjected to trans-aortic constriction (TAC) surgery or sham surgery and Cre activity was induced by intraperitoneal injection followed by feeding with tamoxifen. Mice were sacrificed 4 weeks after TAC or sham surgery. B , Representative photographs of hearts from fibroblast-specific Keap1 -deficient ( Keap1 fl/fl , Postn MCM ) and control ( Postn MCM ) mice 4 weeks after TAC or sham surgery. Scale bars: 5 mm. C , Quantitative analysis of heart weight to tibial length ratio. D , Representative echocardiographic M-mode images of left ventricles and ejection fraction (EF), left ventricular (LV) mass and wall thickness. E , Representative photomicrographs of transverse sections stained with Masson’s trichrome and quantification of interstitial fibrosis area. Scale bars: 500 µm. Data are presented as mean±SD. P values were calculated by two-way ANOVA with Tukey’s multiple comparison test (n=8-16 each).

Article Snippet: Membranes were then probed with anti-Nrf2 (CST, 12721S, 1:1000 for WB), anti-Keap1 (MBL, M-224-3, 1:1000 for WB), anti-Smad3 (CST, 9523S, 1:1000 for WB), anti-Avidin HRP (Biolegend, 405103, 1:2500 for WB), anti-HA tag (CST, 2367, 1:1000 for WB) and anti-β-actin (Sigma, 2228, 1:2000 for WB) antibodies followed by incubation with HRP-conjugated secondary antibodies (CST, 7076S/7074S, 1:1000 for WB).

Techniques: Knock-Out, Activity Assay, Injection, Staining, Comparison

Journal: bioRxiv

Article Title: Fibroblast Nrf2 inhibits profibrotic transcription with Ddx54 and mitigates pathological fibrosis in the mouse heart and kidney

doi: 10.1101/2023.11.09.566496

Figure Lengend Snippet: Quantitative PCR analysis of genes associated with fibrosis and heart failure in the whole hearts of fibroblast-specific Keap1 -deficient ( Keap1 fl/fl , Postn MCM ) and control ( Postn MCM ) mice 4 weeks after TAC or sham surgery. Data are presented as mean±SD. P values were calculated by two-way ANOVA with Tukey’s multiple comparison test (n=8-16 each).

Article Snippet: Membranes were then probed with anti-Nrf2 (CST, 12721S, 1:1000 for WB), anti-Keap1 (MBL, M-224-3, 1:1000 for WB), anti-Smad3 (CST, 9523S, 1:1000 for WB), anti-Avidin HRP (Biolegend, 405103, 1:2500 for WB), anti-HA tag (CST, 2367, 1:1000 for WB) and anti-β-actin (Sigma, 2228, 1:2000 for WB) antibodies followed by incubation with HRP-conjugated secondary antibodies (CST, 7076S/7074S, 1:1000 for WB).

Techniques: Real-time Polymerase Chain Reaction, Comparison

Journal: bioRxiv

Article Title: Fibroblast Nrf2 inhibits profibrotic transcription with Ddx54 and mitigates pathological fibrosis in the mouse heart and kidney

doi: 10.1101/2023.11.09.566496

Figure Lengend Snippet: A , Keap1 fl/fl mice were crossed with Myh6 - MerCreMer ( Myh6 MCM ) mice to generate tamoxifen-inducible cardiomyocyte-specific Keap1 knockout mice. Adult 9-week-old male mice were subjected to transaortic constriction (TAC) or sham surgery and Cre activity was induced by intraperitoneal injection followed by feeding with tamoxifen. The mice were sacrificed 4 weeks after TAC surgery. B , Representative photographs of hearts from cardiomyocyte-specific Keap1 -deficient ( Keap1 fl/fl , Myh6 MCM ) and control ( Myh6 MCM ) mice 4 weeks after TAC or sham surgery. Scale bars: 5 mm. C , Quantitative analysis of heart weight to tibial length ratio. D , Representative echocardiographic M-mode images of left ventricle, ejection fraction (EF), left ventricular (LV) mass, and wall thickness. E , Representative photomicrographs of Masson’s trichrome stained transverse sections and quantification of interstitial fibrosis area of TAC-operated hearts. Scale bars: 500 µm. Data are presented as mean±SD. P values were calculated by two-way ANOVA with Tukey’s multiple comparison test (n=7-10 each).

Article Snippet: Membranes were then probed with anti-Nrf2 (CST, 12721S, 1:1000 for WB), anti-Keap1 (MBL, M-224-3, 1:1000 for WB), anti-Smad3 (CST, 9523S, 1:1000 for WB), anti-Avidin HRP (Biolegend, 405103, 1:2500 for WB), anti-HA tag (CST, 2367, 1:1000 for WB) and anti-β-actin (Sigma, 2228, 1:2000 for WB) antibodies followed by incubation with HRP-conjugated secondary antibodies (CST, 7076S/7074S, 1:1000 for WB).

Techniques: Knock-Out, Activity Assay, Injection, Staining, Comparison

Journal: bioRxiv

Article Title: Fibroblast Nrf2 inhibits profibrotic transcription with Ddx54 and mitigates pathological fibrosis in the mouse heart and kidney

doi: 10.1101/2023.11.09.566496

Figure Lengend Snippet: A , Adult 7-week-old male mice were treated with tamoxifen by intraperitoneal injection and subsequent feeding, and then subjected to UUO (right kidney) or sham surgery (left kidney) at 9 weeks of age. Mice were sacrificed 10 days after UUO surgery. B , Representative photographs of kidneys from fibroblast-specific Keap1 -deficient ( Keap1 fl/fl , Postn MCM ) and control ( Postn MCM ) mice 10 days after UUO or sham surgery. Scale bars: 5 mm. C , Quantitative analysis of the ratio of kidney weight to tibial length. D , Representative photomicrographs of Masson’s trichrome stain sagittal sections and quantification of interstitial fibrosis area of kidneys. Scale bars: 500µm. E , Quantitative PCR analysis of representative fibrosis-related genes in whole kidney tissue. Data are presented as mean±SD. P values were calculated by two-way ANOVA with Tukey’s multiple comparison test (n=12 each).

Article Snippet: Membranes were then probed with anti-Nrf2 (CST, 12721S, 1:1000 for WB), anti-Keap1 (MBL, M-224-3, 1:1000 for WB), anti-Smad3 (CST, 9523S, 1:1000 for WB), anti-Avidin HRP (Biolegend, 405103, 1:2500 for WB), anti-HA tag (CST, 2367, 1:1000 for WB) and anti-β-actin (Sigma, 2228, 1:2000 for WB) antibodies followed by incubation with HRP-conjugated secondary antibodies (CST, 7076S/7074S, 1:1000 for WB).

Techniques: Injection, Staining, Real-time Polymerase Chain Reaction, Comparison

Journal: Redox Report : Communications in Free Radical Research

Article Title: Activation of the Nrf2/Keap1 signaling pathway mediates the neuroprotective effect of Perillyl alcohol against cerebral hypoxic-ischemic damage in neonatal rats

doi: 10.1080/13510002.2024.2394714

Figure Lengend Snippet: Effects of POH on Nrf2/Keap1 pathway-related and apoptosis-related proteins on OGD/R-stimulated PC12 cells. (A) Effects of POH and ML385 on cell viability of OGD/R- stimulated PC12 cells (n = 8). (B) Representative images of Nrf2/Keap1 pathway-related and apoptosis-related proteins by western blot. N-Nrf2 means Nrf2 in the nucleus and c-Nrf2 in the cytoplasm. (C-F) Quantitative analysis of protein expressions above (n ≥ 3). (G) Representative fluorescence image of cleaved caspase-3 (green) with DAPI (blue) (n = 4), scale bar = 100μm. The values were represented as the mean ± SD. ∗ P < .05, ∗∗ P < .01 and ∗∗∗ P < .001 vs. the sham group; ### P < .001 vs. the HI group; &&& P < .001 vs. the HI + POH group.

Article Snippet: Primary antibodies: SOD2/MnSOD (AF5144; Affinity, United States), iNOS (ab178945; Abcam, United Kingdom), GPX4 (WH292781; ABclonal, Wuhan, China), TNF-α (ab66579; Abcam, United Kingdom), IL-6 (21865-1-AP; Proteintech, Wuhan, China), GAPDH (10494-1-AP; Proteintech, Wuhan, China), Nrf2 (#12721; Cell Signaling Technology, United States), Keap1 (WL03285; Wanleibio, China), β-actin (#3700; Cell Signaling Technology, United States), HistoneH3 (#4499; Cell Signaling Technology, United States), BAX (ab32503; Abcam, United Kingdom), BCL2 (ab196495; Abcam, United Kingdom), Caspase-8 (WL03426; Wanleibio, China), Caspase-8 p18 (WL00659; Wanleibio, China), Caspase-3/Cleaved Caspase-3 (WL02117; Wanleibio, China), Cytochrome c (#4272; Cell Signaling Technology, United States), MAP2 (17490-1-AP; Proteintech, Wuhan, China), GFAP (16825-1-AP; Proteintech, Wuhan, China).

Techniques: Western Blot, Fluorescence

Journal: Redox Report : Communications in Free Radical Research

Article Title: Activation of the Nrf2/Keap1 signaling pathway mediates the neuroprotective effect of Perillyl alcohol against cerebral hypoxic-ischemic damage in neonatal rats

doi: 10.1080/13510002.2024.2394714

Figure Lengend Snippet: POH-activated Nrf2/Keap1 signaling pathway attenuated oxidative stress and apoptosis after HI brain injury in neonatal rats. (A) HO-1 mRNA expression in brain tissues 24 hours after HI injury, normalized to β-actin (n = 4). (B-L) Protein expressions of the Nrf2/Keap1 pathway-, oxidative stress- and apoptosis-related proteins were revealed by western blot 24 hours after HI injury and quantitative analyses (n ≥ 3). The values were represented as the mean ± SD. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001 and ∗∗∗∗ P < .0001 vs. the sham group; ### P < .001 vs. the HI group.

Article Snippet: Primary antibodies: SOD2/MnSOD (AF5144; Affinity, United States), iNOS (ab178945; Abcam, United Kingdom), GPX4 (WH292781; ABclonal, Wuhan, China), TNF-α (ab66579; Abcam, United Kingdom), IL-6 (21865-1-AP; Proteintech, Wuhan, China), GAPDH (10494-1-AP; Proteintech, Wuhan, China), Nrf2 (#12721; Cell Signaling Technology, United States), Keap1 (WL03285; Wanleibio, China), β-actin (#3700; Cell Signaling Technology, United States), HistoneH3 (#4499; Cell Signaling Technology, United States), BAX (ab32503; Abcam, United Kingdom), BCL2 (ab196495; Abcam, United Kingdom), Caspase-8 (WL03426; Wanleibio, China), Caspase-8 p18 (WL00659; Wanleibio, China), Caspase-3/Cleaved Caspase-3 (WL02117; Wanleibio, China), Cytochrome c (#4272; Cell Signaling Technology, United States), MAP2 (17490-1-AP; Proteintech, Wuhan, China), GFAP (16825-1-AP; Proteintech, Wuhan, China).

Techniques: Expressing, Western Blot

Journal: Redox Report : Communications in Free Radical Research

Article Title: Activation of the Nrf2/Keap1 signaling pathway mediates the neuroprotective effect of Perillyl alcohol against cerebral hypoxic-ischemic damage in neonatal rats

doi: 10.1080/13510002.2024.2394714

Figure Lengend Snippet: Schematic diagram (by Figdraw) illustrates the potential mechanism of perillyl alcohol for its neuroprotective effect. Hypoxic-ischemic injury resulted in the excessive generation of reactive oxygen species (ROS) within nerve cells, thereby inducing oxidative stress, lipid peroxidation, and mitochondrial dysfunction. Concurrently, hypoxic-ischemic injury triggered reactive astrogliosis in rat brain tissue, ultimately culminating in nerve cell apoptosis. The administration of perillyl alcohol impeded the binding of Nrf2 and Keap1 within nerve cells, thereby stimulating the up-regulation of Nrf2 and its nuclear translocation. Nrf2 subsequently binded to the antioxidant response element (ARE) within the nucleus, thereby promoting the up-regulation of downstream antioxidant/phase II detoxifying enzymes, ultimately mitigating the oxidative stress induced by hypoxic-ischemic injury. The attenuation of oxidative stress modulated the BCL2 family proteins, thereby inhibiting the occurrence of mitochondrial dysfunction and preventing the release of cytochrome C (CytC) from the mitochondria, ultimately blocking the intrinsic apoptosis. Additionally, perillyl alcohol demonstrated the ability to impede reactive astrogliosis in brain tissue and the release of death factors, including TNF-α, by activated astrocytes. This action prevented the activation of death receptors on nerve cell membranes, inhibited the activation of caspase8, and ultimately prevented the occurrence of exogenous apoptosis.

Article Snippet: Primary antibodies: SOD2/MnSOD (AF5144; Affinity, United States), iNOS (ab178945; Abcam, United Kingdom), GPX4 (WH292781; ABclonal, Wuhan, China), TNF-α (ab66579; Abcam, United Kingdom), IL-6 (21865-1-AP; Proteintech, Wuhan, China), GAPDH (10494-1-AP; Proteintech, Wuhan, China), Nrf2 (#12721; Cell Signaling Technology, United States), Keap1 (WL03285; Wanleibio, China), β-actin (#3700; Cell Signaling Technology, United States), HistoneH3 (#4499; Cell Signaling Technology, United States), BAX (ab32503; Abcam, United Kingdom), BCL2 (ab196495; Abcam, United Kingdom), Caspase-8 (WL03426; Wanleibio, China), Caspase-8 p18 (WL00659; Wanleibio, China), Caspase-3/Cleaved Caspase-3 (WL02117; Wanleibio, China), Cytochrome c (#4272; Cell Signaling Technology, United States), MAP2 (17490-1-AP; Proteintech, Wuhan, China), GFAP (16825-1-AP; Proteintech, Wuhan, China).

Techniques: Binding Assay, Translocation Assay, Blocking Assay, Activation Assay