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 Medical Sciences

Article Title: Recombinant Klotho attenuates IFNγ receptor signaling and SAMHD1 expression through blocking NF-κB translocation in glomerular mesangial cells

doi: 10.7150/ijms.78279

Figure Lengend Snippet: Recombinant Klotho protein inhibited IFNγ-induced SAMHD1 protein expression in MES-13 cells. In (A), MES-13 cells were pretreated with recombinant Klotho protein (0.4 nM, 1 nM) for 24 h and then stimulated with 10 ng/mL IFNγ for 24 h. The SAMHD1 protein level in the IFNγ (10 ng/mL)-treated MES-13 cells was used as the control value and set to 100%. The relative protein levels were quantified through scanning densitometry and are expressed as a percentage of the maximal band intensity of the SAMHD1 protein from cultures treated with IFNγ (10 ng/mL). Data are presented as the mean ± SD of Klotho/GAPDH from at least three separate experiments. Asterisks indicate a significant difference from treatment without IFNγ. In (B), the effect of recombinant Klotho protein and PDTC on NFκB nuclear translocation in IFNγ-activated MES-13 cells was visualized through immunofluorescence microscopy. MES-13 cells were pretreated with recombinant Klotho protein (1 nM) for 24 h or with NFκB inhibitor PDTC (10 μM) for 30 min and then stimulated with 10 ng/mL IFNγ for 1.5 h. After fixation on slide glasses, nuclei of MES-13 cells were stained with DAPI to label nuclear DNA (top panels, blue signal). Samples were also stained with an NFκB antibody, and this was followed by signal amplification with a fluorescein isothiocyanate-conjugated secondary antibody (middle panels, green signal). The bottom panel is a merged image of the two upper panels. In (C), the levels of NFκB p65 in nuclear extracts were prepared by nuclear and cytoplasmic extraction as described in methods. Western blots were performed to detect the NFκB distribution in nuclear fractions. The protein amount was quantified by measuring the NFκB bands using densitometric analysis with AlphaEaseFC (Genetic Technologies, Miami, FL, USA) and normalized to histone H3 (H3). All experiments were performed at least in triplicate and reported as a percentage of the control. Data are presented as the mean ± SD of NFκB/H3 derived from at least three separate experiments. Asterisks indicate a significant difference from treatment with IFNγ (10 ng/mL) (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The antibody for SAMHD1 (ab128107) and Klotho (ab203576) and recombinant human Klotho protein (ab84072) were purchased from Abcam (Cambridge, MA).

Techniques: Recombinant, Expressing, Control, Translocation Assay, Immunofluorescence, Microscopy, Staining, Amplification, Extraction, Western Blot, Derivative Assay

Journal: International Journal of Medical Sciences

Article Title: Recombinant Klotho attenuates IFNγ receptor signaling and SAMHD1 expression through blocking NF-κB translocation in glomerular mesangial cells

doi: 10.7150/ijms.78279

Figure Lengend Snippet: Recombinant Klotho protein inhibited IFNγ-induced SAMHD1 expression not through JAK-STAT1 signaling. MES-13 cells were pretreated with recombinant Klotho protein (0.4 nM, 1 nM) for 24 h and then stimulated with 10 ng/mL IFNγ for 1.5 h. The effects of recombinant Klotho protein on IFNγ-induced protein levels of STAT1 and phosphorylated STAT1 (Tyr 701) in MES-13 cells were then determined through Western blot analysis. Phosphorylation of Stat1 at Tyr701 induces transcription factor activation in response to IFNγ. The values of the STAT1 and phosphorylated STAT1 protein levels in IFNγ (10 ng/mL)-treated MES-13 cells were used as controls and set to 100%. Relative protein levels were quantified through scanning densitometry and are expressed as a percentage of the maximal band intensity of the STAT1 and phosphorylated STAT1 proteins from cultures treated with IFNγ (10 ng/mL). Data are presented as the mean ± SD of STAT1 and phosphorylated STAT1 protein/GAPDH derived from at least three separate experiments. Asterisks indicate a significant difference from treatment without IFNγ (∗∗∗∗ p < 0.0001).

Article Snippet: The antibody for SAMHD1 (ab128107) and Klotho (ab203576) and recombinant human Klotho protein (ab84072) were purchased from Abcam (Cambridge, MA).

Techniques: Recombinant, Expressing, Western Blot, Phospho-proteomics, Activation Assay, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: FGF23 Suppresses Chondrocyte Proliferation in the Presence of Soluble α-Klotho both in Vitro and in Vivo

doi: 10.1074/jbc.m112.410043

Figure Lengend Snippet: FIGURE 2. FGF23 makes a protein complex with sKL. A, HEK293 cells were transfected with FGF23-cMyc or sKL-V5 expression vectors. Immunoprecipitation with V5 was followed by Western blot analysis for cMyc. B, conditioned-media containing FGF23-cMyc were mixed with sKL-containing conditioned-media. Immunoprecipitation with cMyc was followed by Western blot analysis for Klotho (AF1819). C, 300 ng of recombinant FGF23 and recombinant sKL were mixed in one milliliter solution and immunoprecipitation with Klotho (Mink1) was followed by Western blot analysis for Klotho (KM2076). D, HEK293 cells were transfected with FGF23-cMyc, and full-length (sKL-FL-V5) or truncated sKL (sKL-KL1-V5 or sKL-KL2-V5) expression vectors. Immunoprecipitation with cMyc was followed by Western blot analysis for V5. The figures shown are the representative from at least three independent experiments.

Article Snippet: Recombinant mouse soluble -Klotho and rat anti-Klotho antibody (AF1819) were purchased from R&D Systems.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Recombinant

Journal: PLoS ONE

Article Title: Klotho Suppresses Cardiomyocyte Apoptosis in Mice with Stress-Induced Cardiac Injury via Downregulation of Endoplasmic Reticulum Stress

doi: 10.1371/journal.pone.0082968

Figure Lengend Snippet: (A,B,C) Quantitative plots of GRP78, HSP47 and CHOP mRNA in CON, ISO and ISO+KL groups for 2, 5 and 9 days. Klotho decreased the GRP78 and HSP47 mRNA from day2 to day9, and it significantly reduced CHOP mRNA at day 9. Data are mean±SEM, n=3. * P<0.05 between two compared groups; NS, no significance.

Article Snippet: Recombinant mouse klotho protein was purchased from R&D system (Minneapolis, MN).

Techniques:

Journal: PLoS ONE

Article Title: Klotho Suppresses Cardiomyocyte Apoptosis in Mice with Stress-Induced Cardiac Injury via Downregulation of Endoplasmic Reticulum Stress

doi: 10.1371/journal.pone.0082968

Figure Lengend Snippet: (A) Representative images of apoptosis assay in H9c2 cells treated by different concentrations of ISO. (B) Demonstration of apoptosis rate.(C) Representative images of apoptosis assay in H9c2 cells treated by ISO and/or klotho. (D) Demonstration of apoptosis rate. Data are mean±SEM, n=5. * P<0.05 between two compared groups; NS, no significance. (E) Representative images of DCF fluorescence signal assessed by FCS Express. (F) Demonstration of the relative DCF fluorescence intensity normalized by CON. Data are mean±SEM. n=3 * P<0.05 between two compared groups; NS, no significance.

Article Snippet: Recombinant mouse klotho protein was purchased from R&D system (Minneapolis, MN).

Techniques: Apoptosis Assay, Fluorescence

Journal: PLoS ONE

Article Title: Klotho Suppresses Cardiomyocyte Apoptosis in Mice with Stress-Induced Cardiac Injury via Downregulation of Endoplasmic Reticulum Stress

doi: 10.1371/journal.pone.0082968

Figure Lengend Snippet: (A) Representative images of western blotting of p-ERK1/2, ERK1/2, p-p38, p38, p-JNK, JNK and GAPDH in H9c2 cells treated by ISO and/or different concentrations of klotho (0.01, 0.1, 1, 10ug/ml). (B,C,D) Demonstration of the phosphorylation of p-ERK1/2(B), p-p38(C) and p-JNK(D) in ISO, ISO+KL(0.01ug/ml), ISO+KL(0.1ug/ml), ISO+KL(1ug/ml), ISO+KL(10ug/ml) groups. Data are mean±SEM, n=3. * P<0.05 between two compared groups; NS, no significance.

Article Snippet: Recombinant mouse klotho protein was purchased from R&D system (Minneapolis, MN).

Techniques: Western Blot, Phospho-proteomics