Journal: ACS Omega

Article Title: Rutin Promotes Wound Healing by Inhibiting Oxidative Stress and Inflammation in Metformin-Controlled Diabetes in Rats

doi: 10.1021/acsomega.3c05595

Figure Lengend Snippet: Topical preparation of rutin-NP downregulated Keap-1 in STZ-induced diabetic rats. Photomicrographs displayed the immunohistochemical reaction of Keap-1 antibody in skin tissues at 21 days among experimental groups stained with Keap-1 antibody (magnification power = 200×, scale bar = 100 μm): (a) The skin section from the untreated control group showed strong expression of Keap-1 antibody (+++) with cytoplasmic and nuclear reactivity along the epidermis and dermis layers (arrows). (b) The skin section from the rutin-NP-treated group exhibited moderate expression of Keap-1 antibody (++) with cytoplasmic and nuclear reactivity of the epidermis and dermis layers (arrows), with a significant difference from the negative control group. (c) The skin section from the β-sitosterol (standard)-treated group indicated low expression of Keap-1 antibody (+) with cytoplasmic and nuclear reactivity along the epidermis and dermis layers (arrows) with a significant difference from the untreated group and rutin-NP group.

Article Snippet: Following overnight incubation at 4 °C with primary anti-Nrf2 antibody, dilution 1:100 (supplied by Biospes, Chongqing, China), primary anti-Keap-1 antibody, dilution 1:100 (supplied by Biospes, Chongqing, China), and primary anti- K i -67 antibody, dilution 1:100 (supplied by Biospes, Chongqing, China), the sections were treated for 1 h with HRP-conjugated goat antirabbit secondary antibody before visualization using a DAB kit.

Techniques: Immunohistochemical staining, Staining, Control, Expressing, Negative Control

Journal: ACS Omega

Article Title: Rutin Promotes Wound Healing by Inhibiting Oxidative Stress and Inflammation in Metformin-Controlled Diabetes in Rats

doi: 10.1021/acsomega.3c05595

Figure Lengend Snippet: Molecular docking of rutin with Keap-1: (A) two-dimensional (2D) rutin–Keap-1 interaction, and (B) three-dimensional (3D) view of rutin in the active site of Keap-1.

Article Snippet: Following overnight incubation at 4 °C with primary anti-Nrf2 antibody, dilution 1:100 (supplied by Biospes, Chongqing, China), primary anti-Keap-1 antibody, dilution 1:100 (supplied by Biospes, Chongqing, China), and primary anti- K i -67 antibody, dilution 1:100 (supplied by Biospes, Chongqing, China), the sections were treated for 1 h with HRP-conjugated goat antirabbit secondary antibody before visualization using a DAB kit.

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 , 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