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: Concanavalin A coated magnetic beads (Bangs Laboratories) were prepared as described and 10 µL of activated beads were added per sample and incubated at RT for 15 min. 10 µL of Concanavalin A coated magnetic beads (Bangs Laboratories) were added per sample and incubated at RT for 10 min. After a quick spin to remove liquid, the bead-bound cells were resuspended in 50 µL Antibody Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail; 0.05% Digitonin; 2 mM EDTA; 0.1% BSA) containing a 1:50 dilution of the appropriate primary antibody; mouse anti-Keap1 antibody (M-224-3, MBL) and rabbit anti-Smad3 antibody (9523S, Cell Signaling).

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: Concanavalin A coated magnetic beads (Bangs Laboratories) were prepared as described and 10 µL of activated beads were added per sample and incubated at RT for 15 min. 10 µL of Concanavalin A coated magnetic beads (Bangs Laboratories) were added per sample and incubated at RT for 10 min. After a quick spin to remove liquid, the bead-bound cells were resuspended in 50 µL Antibody Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail; 0.05% Digitonin; 2 mM EDTA; 0.1% BSA) containing a 1:50 dilution of the appropriate primary antibody; mouse anti-Keap1 antibody (M-224-3, MBL) and rabbit anti-Smad3 antibody (9523S, Cell Signaling).

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: Concanavalin A coated magnetic beads (Bangs Laboratories) were prepared as described and 10 µL of activated beads were added per sample and incubated at RT for 15 min. 10 µL of Concanavalin A coated magnetic beads (Bangs Laboratories) were added per sample and incubated at RT for 10 min. After a quick spin to remove liquid, the bead-bound cells were resuspended in 50 µL Antibody Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail; 0.05% Digitonin; 2 mM EDTA; 0.1% BSA) containing a 1:50 dilution of the appropriate primary antibody; mouse anti-Keap1 antibody (M-224-3, MBL) and rabbit anti-Smad3 antibody (9523S, Cell Signaling).

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: Concanavalin A coated magnetic beads (Bangs Laboratories) were prepared as described and 10 µL of activated beads were added per sample and incubated at RT for 15 min. 10 µL of Concanavalin A coated magnetic beads (Bangs Laboratories) were added per sample and incubated at RT for 10 min. After a quick spin to remove liquid, the bead-bound cells were resuspended in 50 µL Antibody Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail; 0.05% Digitonin; 2 mM EDTA; 0.1% BSA) containing a 1:50 dilution of the appropriate primary antibody; mouse anti-Keap1 antibody (M-224-3, MBL) and rabbit anti-Smad3 antibody (9523S, Cell Signaling).

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: Concanavalin A coated magnetic beads (Bangs Laboratories) were prepared as described and 10 µL of activated beads were added per sample and incubated at RT for 15 min. 10 µL of Concanavalin A coated magnetic beads (Bangs Laboratories) were added per sample and incubated at RT for 10 min. After a quick spin to remove liquid, the bead-bound cells were resuspended in 50 µL Antibody Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail; 0.05% Digitonin; 2 mM EDTA; 0.1% BSA) containing a 1:50 dilution of the appropriate primary antibody; mouse anti-Keap1 antibody (M-224-3, MBL) and rabbit anti-Smad3 antibody (9523S, Cell Signaling).

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: Concanavalin A coated magnetic beads (Bangs Laboratories) were prepared as described and 10 µL of activated beads were added per sample and incubated at RT for 15 min. 10 µL of Concanavalin A coated magnetic beads (Bangs Laboratories) were added per sample and incubated at RT for 10 min. After a quick spin to remove liquid, the bead-bound cells were resuspended in 50 µL Antibody Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail; 0.05% Digitonin; 2 mM EDTA; 0.1% BSA) containing a 1:50 dilution of the appropriate primary antibody; mouse anti-Keap1 antibody (M-224-3, MBL) and rabbit anti-Smad3 antibody (9523S, Cell Signaling).

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: Concanavalin A coated magnetic beads (Bangs Laboratories) were prepared as described and 10 µL of activated beads were added per sample and incubated at RT for 15 min. 10 µL of Concanavalin A coated magnetic beads (Bangs Laboratories) were added per sample and incubated at RT for 10 min. After a quick spin to remove liquid, the bead-bound cells were resuspended in 50 µL Antibody Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail; 0.05% Digitonin; 2 mM EDTA; 0.1% BSA) containing a 1:50 dilution of the appropriate primary antibody; mouse anti-Keap1 antibody (M-224-3, MBL) and rabbit anti-Smad3 antibody (9523S, Cell Signaling).

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: Concanavalin A coated magnetic beads (Bangs Laboratories) were prepared as described and 10 µL of activated beads were added per sample and incubated at RT for 15 min. 10 µL of Concanavalin A coated magnetic beads (Bangs Laboratories) were added per sample and incubated at RT for 10 min. After a quick spin to remove liquid, the bead-bound cells were resuspended in 50 µL Antibody Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail; 0.05% Digitonin; 2 mM EDTA; 0.1% BSA) containing a 1:50 dilution of the appropriate primary antibody; mouse anti-Keap1 antibody (M-224-3, MBL) and rabbit anti-Smad3 antibody (9523S, Cell Signaling).

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

Journal: Burns & Trauma

Article Title: Fibroblast exosomes promote wound healing and improve the quality of healed skin via miR-29a-3p-mediated KEAP1/Nrf2 pathway activation

doi: 10.1093/burnst/tkaf035

Figure Lengend Snippet: Fibroblast exosomes improve wound healing quality by reducing oxidative stress damage. ( a ) Representative images of DHE staining and statistical analysis of the fluorescence intensity of fresh frozen tissue sections from wounds in the control, GW4869 and GW4869 + Fb-exo groups on Days 7 and 28 post-wounding (scale bar = 50 μm, n = 5). ( b ) Statistical analysis of the MDA concentration in wound tissue from the control, GW4869 and GW4869 + Fb-exo groups on Days 7 and 28 post-wounding (n = 5). ( c ) Statistical analysis of the ratio of GSH/GSSG in wound tissue in the control, GW4869 and GW4869 + Fb-exo group on Days 7 and 28 post-wounding (n = 5). ( d ) Representative images of protein immunoblot bands of KEAP1 and Nrf2 in epidermal stem cells and statistical analysis (n = 3). ( e ) Statistical analysis of the MDA concentration and the GSH/GSSG ratio after epidermal stem cells were stimulated with fibroblast exosomes under oxidative stress conditions (n = 3). ( f ) Statistical analysis of cell viability after epidermal stem cells were stimulated with fibroblast exosomes under oxidative stress conditions (n = 3). ( g ) Representative flow cytometry images and statistical analysis of cell apoptosis detected by Annexin V/PI double staining of epidermal stem cells after stimulation with fibroblast exosomes under oxidative stress conditions (n = 3). Mean ± standard deviation ( * P <0.05; * * P < 0.01; * * * P <0.001; * * * * P < 0.0001; ns not statistically significant). DAPI 4′,6-Diamidino-2-phenylindole, DHE dihydroethidium, MDA malondialdehyde, GSH glutathione, GSSG glutathione Disulfide, Nrf2 nuclear factor erythroid 2-related factor 2, KEAP1 Kelch-like ECH-associated protein 1, GAPDH glyceraldehyde-3-phosphate dehydrogenase, CCK8 cell counting kit-8, PI propidium iodide

Article Snippet: Anti-KEAP1 antibody , , HuaBio , Cat. No. HA721525.

Techniques: Staining, Fluorescence, Control, Concentration Assay, Western Blot, Flow Cytometry, Double Staining, Standard Deviation, Cell Counting

Journal: Burns & Trauma

Article Title: Fibroblast exosomes promote wound healing and improve the quality of healed skin via miR-29a-3p-mediated KEAP1/Nrf2 pathway activation

doi: 10.1093/burnst/tkaf035

Figure Lengend Snippet: Fibroblast exosomes increase the capacity for cellular antioxidant stress by suppressing KEAP1 gene transcription via miR-29a-3p. ( a ) Enrichment heatmap of miRs associated with oxidative stress pathways in fibroblast-derived exosomes. ( b ) Intracellular miR-29a-3p levels in epidermal stem cells following the phagocytosis of fibroblast-derived exosomes (n = 3). ( c ) Intracellular KEAP1 mRNA expression levels in epidermal stem cells following phagocytosis of fibroblast-derived exosomes (n = 3). ( d ) Representative western blot analysis images of Nrf2 degradation in response to recombinant KEAP1 protein and fibroblast exosomes (n = 3). ( e ) Prediction of miR-29a-3p binding sites and measurement of relative luciferase activity in epidermal stem cells cotransfected with the miR-29a-3p mimic or negative control mimic and WT or Mut Keap1 vectors (n = 3). ( * P < 0.05; * * P < 0.01; * * * P < 0.001; * * * * P <0.0001; ns not statistically significant). Nrf2 nuclear factor erythroid 2-related factor 2, KEAP1 Kelch-like ECH-associated protein 1, GAPDH glyceraldehyde-3-phosphate dehydrogenase, MUT mutant

Article Snippet: Anti-KEAP1 antibody , , HuaBio , Cat. No. HA721525.

Techniques: Derivative Assay, Expressing, Western Blot, Recombinant, Binding Assay, Luciferase, Activity Assay, Negative Control, Mutagenesis

Journal: Frontiers in immunology

Article Title: Juglone Suppresses Inflammation and Oxidative Stress in Colitis Mice.

doi: 10.3389/fimmu.2021.674341

Figure Lengend Snippet: FIGURE 5 | WB for analysis of the DSS-induced oxidative damage. Colonic p-P65, P65, Keap1 Ucp2, and Nrf2 expression on the protein level were analyzed by WB. (A) Representative western blot images of the colonic p-P65, P65, Keap1, Ucp2, NRF2, and b-Actin proteins. (B–E) Relative densitometric of Ucp2, p-P65/ P65, Keap1, and Nrf2 in the colon. ****P < 0.0001, ##P < 0.01, ###P < 0.001, ####P < 0.0001.

Article Snippet: Furthermore, the tissue slide was treated with 3%H2O2 for 25 min and incubated with primary antibodies against UCP1, Nrf2, Keap1 (1:100; Servicebio Technology Co. Ltd., Wuhan, China) at 4°C overnight.

Techniques: Expressing, Western Blot

Journal: Frontiers in immunology

Article Title: Juglone Suppresses Inflammation and Oxidative Stress in Colitis Mice.

doi: 10.3389/fimmu.2021.674341

Figure Lengend Snippet: FIGURE 6 | Immunofluorescence staining for analysis of the DSS-induced oxidative damage. Immunofluorescence staining was assigned to measure the colonic expression of Ucp2 (A), Keap1 (B), and Nrf2 (C). * meant P < 0.05, and # meant 0.1 < P < 0.05, vs. the DSS group.

Article Snippet: Furthermore, the tissue slide was treated with 3%H2O2 for 25 min and incubated with primary antibodies against UCP1, Nrf2, Keap1 (1:100; Servicebio Technology Co. Ltd., Wuhan, China) at 4°C overnight.

Techniques: Staining, Expressing