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: Cell communication and signaling : CCS

Article Title: FGF19 increases mitochondrial biogenesis and fusion in chondrocytes via the AMPKα-p38/MAPK pathway.

doi: 10.1186/s12964-023-01069-5

Figure Lengend Snippet: Fig. 1 FGF19 induces a transient increase in mitochondrial number and an enhanced generation of ATP products. a Representative TEM images showing the changes of mitochondrial number in chondrocytes induced by FGF19 at 200 ng/ml in the presence of KLB (200 ng/ml). The images were chosen based on three independent experiments (n = 3). Orange arrows indicated individual mitochondrion. b Quantification of mitochondrial number (per cell) in chondrocytes induced by FGF19 at 200 ng/ml in the presence of KLB (200 ng/ml). Quantitative analyses of the mitochondrial number were based on nine cells (per group) from three independent experiments (n = 3). c Representative immunofluorescent staining showing the number changes of mitochondria in living chondrocytes induced by FGF19 (200 ng/ml) in the presence of KLB (200 ng/ml) for 72 h. The images were chosen based on three independent experiments (n = 3). Red, individual mitochondrion; Green, F-actin; Blue, nucleus. d Linear quantification of fluorescence intensity of mitochondrion number in chondrocytes induced by FGF19 at 200 ng/ml in the presence of KLB (200 ng/ml) by Image Pro Plus 6.0. e ATP assay showing the increase of intracellular ATP products in chondrocytes induced by FGF19 at 200 ng/ml in the presence of KLB (200 ng/ml). The results were based on three independent experiments (n = 3). f Representative western blotting showing the expression change of CS in chondrocytes induced by FGF19 at 200 ng/ml in the presence of KLB (200 ng/ml). The images were chosen based on three independent experiments (n = 3). g Quantification of CS by western blotting in (f). The data in b are shown as box (from 25, 50 to 75%) and whisker (minimum to maximum values) plots. The significant difference analysis in b, e and g was based on Student T-test

Article Snippet: FGF19 (200 ng/ml, No.100-32, PEPRO TECH, USA) and/or KLB (200 ng/ ml, 2619-KB-050, R&D Systems, USA) at a 1:1 ratio were added into the culture media as the experimental group and continued to incubate for 72 h. Thaw all the components of Cell NavigatorTM Mitochondrion Staining Kit at RT before starting the experiment.

Techniques: Staining, Fluorescence, ATP Assay, Western Blot, Expressing, Whisker Assay

Journal: Cell communication and signaling : CCS

Article Title: FGF19 increases mitochondrial biogenesis and fusion in chondrocytes via the AMPKα-p38/MAPK pathway.

doi: 10.1186/s12964-023-01069-5

Figure Lengend Snippet: Fig. 4 FGF19 activates p38/MAPK signalling in chondrocytes. a RNA sequencing showing the changes in the expression of MAPK-related mediators in chondrocytes induced by FGF19 at 200 ng/ml in the presence of KLB (200 ng/ml). Three pairs of samples were obtained from three independent cell isolates (n = 3), namely, samples 1, 1′, 1′′and 1′′′, samples 2, 2′, 2′′and 2′′′, and samples 3, 3′, 3′′and 3′′′. The data were present as log2(FPKM + 1). b Representative western blotting showing the expression change of ERK, p-ERK, p38, p-p38, JNK and p-JNK in chondrocytes induced by FGF19 at 200 ng/ml in the presence of KLB (200 ng/ml). The images were chosen based on three independent experiments (n = 3). c Quantification of p38 and p-p38 by western blotting in (b). d Representative immunofluorescent staining showing the change in the expression and distribution of p-p38 in chondrocytes induced by FGF19 (200 ng/ml) in the presence of KLB (200 ng/ml) for 72 h. The images were chosen based on three independent experiments (n = 3). Red, p-p38; Green, F-actin; Blue, nucleus. e Quantification of fluorescence intensity of p-p38 in chondrocytes induced by FGF19 at 200 ng/ml in the presence of KLB (200 ng/ml). The data were based on nine cells from three independent experiments. The data in e were shown as box (from 25, 50 to 75%) and whisker (minimum to maximum values) plots. The significant difference analysis in c and e was based on Student T-test

Article Snippet: FGF19 (200 ng/ml, No.100-32, PEPRO TECH, USA) and/or KLB (200 ng/ ml, 2619-KB-050, R&D Systems, USA) at a 1:1 ratio were added into the culture media as the experimental group and continued to incubate for 72 h. Thaw all the components of Cell NavigatorTM Mitochondrion Staining Kit at RT before starting the experiment.

Techniques: RNA Sequencing, Expressing, Western Blot, Staining, Fluorescence, Whisker Assay

Journal: Cell communication and signaling : CCS

Article Title: FGF19 increases mitochondrial biogenesis and fusion in chondrocytes via the AMPKα-p38/MAPK pathway.

doi: 10.1186/s12964-023-01069-5

Figure Lengend Snippet: Fig. 5 Inhibition of p38 attenuated FGF19-enhanced AMPKα activity. a Representative western blotting showing the expression change of p38, p-p38, AMPKα, p-AMPKα, PGC-1α and SIRT1 in chondrocytes induced by SB203580 (10 µM) in the presence of FGF19 (200 ng/ml) and KLB (200 ng/ml). The images were chosen based on three independent experiments (n = 3). b Quantification of p38, p-p38, AMPKα, p-AMPKα, PGC-1α and SIRT1 by western blotting in (a). c Representative immunofluorescent staining showing the change in the distribution of p-AMPKα in chondrocytes induced by SB203580 (10 µM) in the presence of FGF19 (200 ng/ml) and KLB (200 ng/ml). The images were chosen based on three independent experiments (n = 3). Red, p-AMPKα; Green, F-actin; Blue, nucleus. d Representative immunofluorescent staining showing the change in the expression and distribution of PGC-1α in chondrocytes induced by SB203580 (10 µM) in the presence of FGF19 (200 ng/ml) and KLB (200 ng/ml). The images were chosen based on three independent experiments (n = 3). Red, PGC-1α; Green, F-actin; Blue, nucleus. e Quantification of fluorescence intensity of p-AMPKα and PGC-1α in chondrocytes induced by SB203580 (10 µM) in the presence of FGF19 (200 ng/ml) and KLB (200 ng/ml). The data in e were shown as box (from 25, 50 to 75%) and whisker (minimum to maximum values) plots. The significant difference analysis in b and e was based on Student T-test

Article Snippet: FGF19 (200 ng/ml, No.100-32, PEPRO TECH, USA) and/or KLB (200 ng/ ml, 2619-KB-050, R&D Systems, USA) at a 1:1 ratio were added into the culture media as the experimental group and continued to incubate for 72 h. Thaw all the components of Cell NavigatorTM Mitochondrion Staining Kit at RT before starting the experiment.

Techniques: Inhibition, Activity Assay, Western Blot, Expressing, Staining, Fluorescence, Whisker Assay

Journal: The Journal of Neuroscience

Article Title: The Antiaging Protein Klotho Enhances Oligodendrocyte Maturation and Myelination of the CNS

doi: 10.1523/jneurosci.2080-12.2013

Figure Lengend Snippet: Figure 1. Phosphorylation kinetic analysis of Klotho-treated OPCs. A, OPCs were treated with Klotho for the times indicated, and the cell lysates were analyzed by Western blotting for protein phosphorylation of the proteins indicated. B, Kinetics of the signal intensity based on band densitometry as in A. Results represent the average of two independent experiments. Phosphory- lated ERK1/2 and Akt are normalized to total ERK1/2 and Akt, respectively. All other phosphorylated proteins are normalized to GAPDH internal control.

Article Snippet: The recombinant mouse Klotho protein containing the extracellular domain of mouse Klotho (Ala 35-Lys 982) was from R&D Systems.

Techniques: Phospho-proteomics, Western Blot, Control

Journal: The Journal of Neuroscience

Article Title: The Antiaging Protein Klotho Enhances Oligodendrocyte Maturation and Myelination of the CNS

doi: 10.1523/jneurosci.2080-12.2013

Figure Lengend Snippet: Figure 2. Klotho effects in vitro on primary OPCs. A–D, Klotho enhances oligodendrocyte maturation. Rat OPCs were treated with Klotho ( Klotho) or with PBS (Control) for 3 d, and then immunostainedforthematureoligodendrocytemarkerO1(green),andpan-oligodendrocytemarkerOlig2(red).A,AtypicalO1stainingofadifferentiatedoligodendrocyte.B,Statisticalanalysis ofO1andOlig2stainingofOPCsat3and6dtreatmentwithKlothoorPBS.*Statisticalsignificance(p0.005,ttest).ErrorbarsindicateSD.C,ImmunostainingofOPCswithantibodiestothemature oligodendrocytemarkerCC-1(green)andpan-oligodendrocytemarkerOlig2(red).CellnucleiwerestainedwithDAPI(blue).Whitearrowsinthemergedimageindicatenonoligodendrocyticcells, andyellowarrowsindicateundifferentiatedoligodendrocytes.D,StatisticalanalysisoftheratioofCC-1toOlig2stainingofOPCsat3,6,and8doftreatmentwithKlothoorPBSinmedium1(OPC culturemediumcontainingCNTFandT3)ormedium2(OPCculturemediumcontainingCNTF,T3,andNT3).*Statisticalsignificance(p0.005,ttest).ErrorbarsindicateSD.E,KlothoenhancesOPC maturationviaERKandAktsignaling.RatOPCsweretreatedwith0.5MLY294002(LY)(forAktinhibition)or1MUO126(UO)(forERKinhibition)for30minbeforeKlotho(KL)wasadded.OPCs weretreatedfor3dandthenimmunostainedasinD.Statisticalanalysisoftheresultsisplotted.F,KlothoenhancesmajormyelinproteinsexpressioninratOPCs.OPCsweretreatedwithorwithout Klothofor6dinculturemediumcontainingCNTFandT3andblottedwiththeantibodiesindicatedwithtubulinasloadingcontrol.G,Therelativesignalintensitybasedonbanddensitometrywas plottedasrelativepercentagetocontrolwithoutKlothotreatment.Resultsrepresenttheaverageofthreetofiveindependentexperiments.Allproteinsarenormalizedtotubulininternalcontrol. *Statisticalsignificance(p0.005,ttest).ErrorbarsindicateSD.H,KlothodoesnotaffectOPCcellviability.CellTiterGloassaywasusedtoassessKlotho’seffectonOPCcellviabilityafter1–4dof Klotho treatment. Error bars indicate SD. Results represent the average of six independent experiments. I, Klotho does not affect OPC cell proliferation. Cell numbers were assayed by crystal violet staining at day 2 and day 4 after Klotho treatment. Error bars indicate SD. Results represent the average of three independent experiments.

Article Snippet: The recombinant mouse Klotho protein containing the extracellular domain of mouse Klotho (Ala 35-Lys 982) was from R&D Systems.

Techniques: In Vitro, Control, Staining

Journal: The Journal of Neuroscience

Article Title: The Antiaging Protein Klotho Enhances Oligodendrocyte Maturation and Myelination of the CNS

doi: 10.1523/jneurosci.2080-12.2013

Figure Lengend Snippet: Figure3. qRT-PCRconfirmationoftheeffectofKlothoonOPCmaturation.A,RepresentativeqPCRresultsofdifferentiatedOPCs for3and7d.ThehousekeepinggeneGAPDHandthetoptwoexpressedgenes,MBPandPlp1,areindicated.B,Foldchangeresults oftheupregulatedgenesbyqPCRanalysisofRNAfromOPCstreatedwithKlothofor7dcomparedwithcontrol.OPCswerecultured inmediumcontainingbFGFandPDGFfor3d,andthenchangedtothedifferentiationmediumwithCNTFandT3withorwithout Klotho for 7 d. *Statistical significance (p 0.05, t test).

Article Snippet: The recombinant mouse Klotho protein containing the extracellular domain of mouse Klotho (Ala 35-Lys 982) was from R&D Systems.

Techniques:

Journal: The Journal of Neuroscience

Article Title: The Antiaging Protein Klotho Enhances Oligodendrocyte Maturation and Myelination of the CNS

doi: 10.1523/jneurosci.2080-12.2013

Figure Lengend Snippet: Figure 4. Klotho knock-out mice exhibit impaired myelination. A–L, The 4.5-week-old Klotho /, Klotho /, or Klotho / mice were processed for electron microscopy. Cross-sectional imagesshowexamplesofmyelinationpatternsforopticnerve(A-F)at2900(A,C,E)and5800(B,D,F)andcorpuscallosum(G–L)at2900(G,I,K)and5800(H,J,L).Scalebars:E,2m; F,500nm.As,Astrocyte.M,Thenumberofmyelinatedandunmyelinatedaxonswerecountedandgraphedaspercentageofmyelinatedfibers.Averagesrepresentaxonalcountsanalyzedfrom3 to6differentimages.*Statisticalsignificance(p0.0001,ttest).ErrorbarsindicateSD.ON,Opticnerve;CC,corpuscallosum;SC,spinalcord.N,KlothoexpressioninCC,SC,andON.Westernblot analysis of Klotho from the lysates of 1.1- and 1.6-month-old control mice CC, SC, and ON tissues. O, Statistical analysis of Klotho expression in N with GAPDH as loading control. *Statistical significance (p 0.05, t test). Error bars indicate SD. Sample size is n 4 for each age. P, Major myelin proteins were largely reduced in Klotho knock-out mice. Western blot analysis of myelin markersfromthebrainlysatesof8-week-oldKlothoknock-out(KL /)andcontrol(KL /)mice.Q,StatisticalanalysisofthemyelinmarkersinPwithtubulinascontrol.*Statisticalsignificance (p 0.05, t test). Error bars indicate SD.

Article Snippet: The recombinant mouse Klotho protein containing the extracellular domain of mouse Klotho (Ala 35-Lys 982) was from R&D Systems.

Techniques: Knock-Out, Electron Microscopy, Control, Expressing, Western Blot

Journal: The Journal of Neuroscience

Article Title: The Antiaging Protein Klotho Enhances Oligodendrocyte Maturation and Myelination of the CNS

doi: 10.1523/jneurosci.2080-12.2013

Figure Lengend Snippet: Figure5. AxonalmicrodomainsarealteredinKlotho /mice.Fivesectionseachofcorpuscallosumfromtwowild-typeand two homozygous Klotho / mice were stained for caspr (green) and -IV spectrin (red). A, C, Wild-type mice have normal compact paranodal and nodal structure. B, D, Klotho / mice show an abundance of shorter paranodal segments and longer nodal segments (arrows). E, Measurements of nodal and paranodal length show a statistically significant decrease in paranodal lengthinKlotho /miceassociatedwithasignificantincreaseinnodallength.*Statisticalsignificance(p0.001,ttest).Scale bar, 2 m. F, Electron microscopic longitudinal section analysis of nodes of Ranvier in corpus callosum from wild-type (KL /) and Klotho / (KL /) mice at 13,000. Arrows indicate the junction between node and paranode. N, Node; P: paranode. Scale bars, 500 nm.

Article Snippet: The recombinant mouse Klotho protein containing the extracellular domain of mouse Klotho (Ala 35-Lys 982) was from R&D Systems.

Techniques: Staining

Journal: The Journal of Neuroscience

Article Title: The Antiaging Protein Klotho Enhances Oligodendrocyte Maturation and Myelination of the CNS

doi: 10.1523/jneurosci.2080-12.2013

Figure Lengend Snippet: Figure 6. Immunohistochemistry and quantitative analysis of expression of Olig2 and GST-Pi in brain sections from 5-week-old Klotho / and Klotho / mice. A–D, IHC images show examples of Olig2 nuclear staining patterns at 4 (A, B) and 20 (C, D). Scale bars: A, B, 200 m; C, D, 50 m. A, C, KL /. B, D, KL /. The fimbria region is outlined. Hb, Habenula. E, Cell countingofOlig2 wasperformedasdescribedinMaterialsandMethods.ThenumberofOlig2 cellsinfimbriawerecountedandthepercentageofOlig2 cellsinKL /andKL /graphed. *Statistical significance (p 0.01, t test). Error bars indicate SD. F–I, IHC of brain sections with antibodies to the mature oligodendrocyte marker GST-Pi at 4 (F, G) and 20 (H, I). Scale bars: F,G,200m;H,I,50m.J,QuantitationofGST-Pi cellsasdescribedinMaterialsandMethods.TheGST-Pi cellsinfimbriawerecountedandthepercentageofGST-Pi cellsinKL /and KL / graphed. *Statistical significance (p 0.05, t test). Error bars indicate SD.

Article Snippet: The recombinant mouse Klotho protein containing the extracellular domain of mouse Klotho (Ala 35-Lys 982) was from R&D Systems.

Techniques: Immunohistochemistry, Expressing, Staining, Marker

Journal: The Journal of Neuroscience

Article Title: The Antiaging Protein Klotho Enhances Oligodendrocyte Maturation and Myelination of the CNS

doi: 10.1523/jneurosci.2080-12.2013

Figure Lengend Snippet: Figure 7. Transcription factors involved in Klotho regulation of OPCs. A, Western blot analysis of STAT3 phosphorylation upon Klotho treatment of OPC cells. B, Luciferase assay of luciferase reporters in OPC cells. OPCs were transfected with the reporter plasmidswithRenillaluciferaseandtreatedwithorwithoutKlothofor24hfollowedbyluciferaseassay.Thefoldchangecompar- ing Klotho treated to control is shown. p values indicate statistical significance by t test.

Article Snippet: The recombinant mouse Klotho protein containing the extracellular domain of mouse Klotho (Ala 35-Lys 982) was from R&D Systems.

Techniques: Western Blot, Phospho-proteomics, Luciferase, Transfection, Control