recombinant murine klotho  (R&D Systems)

Journal: American Journal of Translational Research

Article Title: Klotho mitigates diquat-induced myocardial injury in rats by activating Nrf2/ARE-mediated suppression of oxidative stress

doi: 10.62347/SKBR3572

Figure Lengend Snippet: Klotho alleviated DQ-induced cardiomyocyte oxidative stress. (A, B) Cell viability was examined using Cell Counting Kit-8 (CCK-8) assay. (C) Klotho protein levels were examined using western blot analysis. (D) DQ-stimulated H9c2 cardiomyocytes were treated with recombinant Klotho protein, followed by viability assessment using CCK-8 assay. (E, F) Apoptosis was examined using flow cytometry. (G, H) ROS fluorescence signal, (I) MDA level, (J) GSH-ST, (K) GSH-PX, and (L) SOD activities in H9c2 cells were assessed using ELISA kits. (M, N) Nrf2, HO-1, and NQO1 protein levels were examined using western blot analysis. Data were presented as mean ± SD. * P <0.05, ** P <0.01, compared to control group. ## P <0.01, compared to DQ group.

Article Snippet: To induce oxidative injury, cells were treated with 50 μM DQ (45422, Sigma-Aldrich, USA) for 24 h. For Klotho intervention, cells were treated with 1 μg/mL recombinant Klotho protein (1819-KL, R&D Systems, USA) [ 19 , 20 ].

Techniques: Cell Counting, CCK-8 Assay, Western Blot, Recombinant, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Control

Journal: American Journal of Translational Research

Article Title: Klotho mitigates diquat-induced myocardial injury in rats by activating Nrf2/ARE-mediated suppression of oxidative stress

doi: 10.62347/SKBR3572

Figure Lengend Snippet: Nrf2 inhibition abrogated the protective effects of Klotho on DQ-induced oxidative stress. DQ-stimulated H9c2 cells received recombinant Klotho protein and Nrf2 inhibitor ML385. (A, B) Nrf2, HO-1, and NQO1 protein levels were examined using western blot analysis. (C) Cell viability was examined using CCK-8 assay. (D, E) Apoptosis was examined using flow cytometry. (F, G) ROS fluorescence signal, (H) MDA level, (I) GSH-ST, (J) GSH-PX, and (K) SOD activities in H9c2 cells were assessed using ELISA kits. Data were presented as mean ± SD. ** P <0.01, compared to control group. ## P <0.01, compared to DQ group. && P <0.01, compared to DQ+Klotho group.

Article Snippet: To induce oxidative injury, cells were treated with 50 μM DQ (45422, Sigma-Aldrich, USA) for 24 h. For Klotho intervention, cells were treated with 1 μg/mL recombinant Klotho protein (1819-KL, R&D Systems, USA) [ 19 , 20 ].

Techniques: Inhibition, Recombinant, Western Blot, CCK-8 Assay, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Control

Journal: American Journal of Translational Research

Article Title: Klotho mitigates diquat-induced myocardial injury in rats by activating Nrf2/ARE-mediated suppression of oxidative stress

doi: 10.62347/SKBR3572

Figure Lengend Snippet: Klotho alleviated DQ-induced acute myocardial injury in rats through Nrf2/ARE activation. Rats were intragastrically administered with DQ to induce acute myocardial injury and treated with recombinant Klotho protein for 5 days. (A, B) Klotho protein expression in myocardial tissues was assessed using immunohistochemistry. (C) H&E staining of myocardial tissue of rats in each group (D, E) ROS fluorescence signal, (F) MDA level, (G) GSH-ST, (H) GSH-PX, and (I) SOD activities in H9c2 cells were assessed using ELISA kits. (J, K) Nrf2, HO-1, and NQO1 protein levels in myocardial tissues was assessed using immunohistochemistry. ** P <0.01, compared to control group. ## P <0.01, compared to DQ group.

Article Snippet: To induce oxidative injury, cells were treated with 50 μM DQ (45422, Sigma-Aldrich, USA) for 24 h. For Klotho intervention, cells were treated with 1 μg/mL recombinant Klotho protein (1819-KL, R&D Systems, USA) [ 19 , 20 ].

Techniques: Activation Assay, Recombinant, Expressing, Immunohistochemistry, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Molecular Medicine

Article Title: Klotho attenuates epithelial-mesenchymal transition of retinal pigment epithelial cells in subretinal fibrosis by suppressing the ERK1/2 and Wnt/β-catenin signaling pathways

doi: 10.3892/ijmm.2025.5486

Figure Lengend Snippet: TGF-β1 induces EMT and downregulates klotho expression in ARPE-19 cells. (A) TGF-β1-induced morphological changes in APRE-19 cells. Scale bar, 200 μ m. (B) Expression levels of EMT-associated markers in response to TGF-β1 stimulation were detected by western blotting. (C) Relative protein expression (normalized to GAPDH) in western blot. (D) Cell Counting Kit-8 assay was used to determine the proliferation of ARPE-19 cells treated with 10 ng/ml TGF-β1. (E) EdU assays were used to confirm proliferation of ARPE-19 cells treated with 10 ng/ml TGF-β1 for 24 h. Scale bar, 100 μ m. (F) Quantification of the proliferative ability of cells in EdU assays. (G) Representative migration of ARPE-19 cells treated with TGF-β1. (H) Quantification of the migratory ability of cells in Transwell migration assay. Scale bar, 400 μ m. (I) Wound healing of ARPE-19 cells treated with TGF-β1Scale bar, 200 μ m. (J) Quantification of the migratory ability of cells in scratch assay. (K) Relative mRNA expression (normalized to GAPDH) of klotho in cells treated with or without TGF-β1. (L) Relative expression of klotho in cells treated with or without TGF-β1 were detected by western blotting. (M) Quantification of the relative protein expression (normalized to GAPDH) in western blot. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 vs. control group. EMT, epithelial-mesenchymal transition; SMA, smooth muscle actin; ZO, zona occludens; Cad, cadherin; OD, optical density.

Article Snippet: Recombinant mouse klotho protein (cat. no. 1819-KL, R&D Systems, Inc.) or PBS was administered intravitreally 3 days after photocoagulation.

Techniques: Expressing, Western Blot, Cell Counting, Migration, Transwell Migration Assay, Wound Healing Assay, Control

Journal: International Journal of Molecular Medicine

Article Title: Klotho attenuates epithelial-mesenchymal transition of retinal pigment epithelial cells in subretinal fibrosis by suppressing the ERK1/2 and Wnt/β-catenin signaling pathways

doi: 10.3892/ijmm.2025.5486

Figure Lengend Snippet: Klotho overexpression attenuates EMT in ARPE-19 cells induced by TGF-β1. ARPE-19 cells were infected with Lv-klotho or Lv-NC, followed by treatment with or without TGF-β1. (A) Protein levels of EMT-associated markers were analyzed using western blotting. (B) Relative ZO-1 protein expression (normalized to GAPDH) in western blot. (C) Quantification of the relative α-SMA protein expression (normalized to GAPDH) in western blot. (D) Quantification of the relative N-cad protein expression (normalized to GAPDH) in western blot. (E) Quantification of the relative collagen I protein expression (normalized to GAPDH) in western blot. (F) Cell Counting Kit-8 assays were used to determine the proliferation of ARPE-19 cells (G) EdU assays were used to assess the proliferation of ARPE-19 cells. Scale bar, 100 μ m. (H) Quantification of the proliferative ability of cells in EdU assays. (I) Representative Transwell cell migration assay. Scale bar, 200 μ m. (J) Quantitative analysis of the migratory ability of cells. (K) Wound healing assay was used to assess cell migration. Scale bar, 200 μ m. (L) Quantification of the migratory ability of cells in wound healing assay. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Lv-NC, lentiviral vector negative control; EMT, epithelial-mesenchymal transition; SMA, smooth muscle actin; ZO, zona occludens; Cad, cadherin; OD, optical density.

Article Snippet: Recombinant mouse klotho protein (cat. no. 1819-KL, R&D Systems, Inc.) or PBS was administered intravitreally 3 days after photocoagulation.

Techniques: Over Expression, Infection, Western Blot, Expressing, Cell Counting, Cell Migration Assay, Wound Healing Assay, Migration, Plasmid Preparation, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Klotho attenuates epithelial-mesenchymal transition of retinal pigment epithelial cells in subretinal fibrosis by suppressing the ERK1/2 and Wnt/β-catenin signaling pathways

doi: 10.3892/ijmm.2025.5486

Figure Lengend Snippet: Knockdown of klotho induces EMT in ARPE-19 cells. ARPE-19 cells were infected with lentiviral plasmids harboring sh-klotho or empty vector. (A) Klotho expression was measured using RT-qPCR following klotho overexpression. (B) Protein expression levels of klotho. (C) Quantification of the relative protein expression (normalized to GAPDH) in western blot. (D) Klotho expression was measured using RT-qPCR following knockdown. (E) Protein expression levels of EMT-related proteins were detected following klotho knockdown. (F) Quantification of the relative protein expression (normalized to GAPDH) in western blot. * P<0.05, ** P<0.01, *** P<0.001 vs. sh-NC. sh, short hairpin; RT-q, reverse transcription-quantitative; NC, negative control; EMT, epithelial-mesenchymal transition; SMA, smooth muscle actin; ZO, zona occludens; Cad, cadherin; Lv, lentiviral vector.

Article Snippet: Recombinant mouse klotho protein (cat. no. 1819-KL, R&D Systems, Inc.) or PBS was administered intravitreally 3 days after photocoagulation.

Techniques: Knockdown, Infection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Over Expression, Western Blot, Reverse Transcription, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Klotho attenuates epithelial-mesenchymal transition of retinal pigment epithelial cells in subretinal fibrosis by suppressing the ERK1/2 and Wnt/β-catenin signaling pathways

doi: 10.3892/ijmm.2025.5486

Figure Lengend Snippet: Bioinformatics analysis following klotho overexpression. (A) Principal component analysis and (B) volcano plot of significant DEGs. (C) GO and (D) enrichment of genes and pathways. DEG, differentially expressed gene; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; Lv, lentiviral vector; NC, negative control; FDR, false discovery rate; FC, fold-change; PCA, principal components analysis.

Article Snippet: Recombinant mouse klotho protein (cat. no. 1819-KL, R&D Systems, Inc.) or PBS was administered intravitreally 3 days after photocoagulation.

Techniques: Over Expression, Plasmid Preparation, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Klotho attenuates epithelial-mesenchymal transition of retinal pigment epithelial cells in subretinal fibrosis by suppressing the ERK1/2 and Wnt/β-catenin signaling pathways

doi: 10.3892/ijmm.2025.5486

Figure Lengend Snippet: Klotho regulates EMT of ARPE-19 cells via the ERK1/2 and Wnt/β-catenin signaling pathway. Protein expression levels of (A) ERK signaling pathway were analyzed using western blotting. (B) Relative protein expression. (C) β-catenin pathway were analyzed using western blotting. (D) Quantification of the relative protein expression of β-catenin. (E) Quantification of the relative protein expression of c-Myc. (F) Quantification of the relative protein expression of CyclinD1 (normalized to GAPDH) in western blot. * P<0.05, ** P<0.01, *** P<0.001. EMT, epithelial-mesenchymal transition; Lv, lentiviral vector; NC, negative control; p, phosphorylated.

Article Snippet: Recombinant mouse klotho protein (cat. no. 1819-KL, R&D Systems, Inc.) or PBS was administered intravitreally 3 days after photocoagulation.

Techniques: Expressing, Western Blot, Plasmid Preparation, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Klotho attenuates epithelial-mesenchymal transition of retinal pigment epithelial cells in subretinal fibrosis by suppressing the ERK1/2 and Wnt/β-catenin signaling pathways

doi: 10.3892/ijmm.2025.5486

Figure Lengend Snippet: Klotho alleviates progression of subretinal fibrosis in a mouse model of choroidal neovascularization. (A) Representative confocal images of RPE-choroid-sclera flat-mounts stained for collagen I in mice with subretinal fibrosis treated with 10 or 20 nM klotho. Scale bar, 100 μ m. (B) Quantitative analysis of collagen I-positive lesion area in the mice. (C) Representative Masson staining of collagen fibers (black arrow). Magnification, ×200, Scale bar, 200 μ m. (D) Semiquantitative analyses of the Masson staining showed that subretinal deposition of collagen was alleviated following klotho treatment. (E) Representative confocal images of eye sections stained for RPE65 (green) and α-SMA (red), showing the presence of RPE65 + α-SMA + cells (white arrows) in the fibrotic lesion. Cell nuclei were stained with DAPI (blue). Scale bar, 10 μ m. (F) Western blotting was used to determine protein expression levels of epithelial-mesenchymal transition-related markers. (G) Relative ZO-1 protein expression (normalized to GAPDH) in western blot. (H) Quantification of relative α-SMA protein expression (normalized to GAPDH) in western blot. (I) Quantification of the relative N-cad protein expression (normalized to GAPDH) in western blot. (J) Quantification of the relative collagen I protein expression (normalized to GAPDH) in western blot. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. RPE, retinal pigment epithelium; SMA, smooth muscle actin; ZO, zona occludens; cad, cadherin.

Article Snippet: Recombinant mouse klotho protein (cat. no. 1819-KL, R&D Systems, Inc.) or PBS was administered intravitreally 3 days after photocoagulation.

Techniques: Staining, Western Blot, Expressing

Journal: International Journal of Molecular Medicine

Article Title: Klotho attenuates epithelial-mesenchymal transition of retinal pigment epithelial cells in subretinal fibrosis by suppressing the ERK1/2 and Wnt/β-catenin signaling pathways

doi: 10.3892/ijmm.2025.5486

Figure Lengend Snippet: Klotho alleviates progression of subretinal fibrosis in vivo via ERK1/2 and Wnt/β-catenin signaling pathway. (A) Protein expression levels of ERK pathway were analyzed using western blotting. (B) Quantification of the relative protein expression (normalized to GAPDH) in western blot. (C) β-catenin pathway were analyzed using western blotting. (D) Relative β-catenin protein expression (normalized to GAPDH) in western blot. (E) Quantification of the relative c-Myc protein expression (normalized to GAPDH) in western blot. (F) Quantification of the relative cyclinD1 protein expression (normalized to GAPDH) in western blot. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. p, phosphorylated.

Article Snippet: Recombinant mouse klotho protein (cat. no. 1819-KL, R&D Systems, Inc.) or PBS was administered intravitreally 3 days after photocoagulation.

Techniques: In Vivo, Expressing, Western Blot