Journal: Lipids in Health and Disease

Article Title: Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways

doi: 10.1186/s12944-017-0447-0

Figure Lengend Snippet: Effect of Klotho on cellular viability in HUVECs. Cells were exposed to various concentrations of ox-LDL (25, 50, 100, and 200 μg/ml) for 24 h. a HUVEC morphology was observed under an inverted phase contrast microscope (×10) following 24 h of ox-LDL treatment. Typical cellular fragmentations, vacuoles, and debris are arrowed. (Bar = 50 μm) ( b ) Cellular viability was detected by MTT assay. c and d SOD enzymatic activity and MDA levels in HUVECs were analyzed using commercially available assay kits. e HUVECs were pretreated with 100, 200, 400, and 800 pM of Klotho for 1 h, and then incubated with ox-LDL (50 μg/ml) for 24 h. Cellular viability was detected by MTT assay. Data are shown as mean ± S.D. ( n = 3). Statistical differences are expressed as ## p < 0.01 vs. control, * p < 0.05, ** p < 0.01 vs. ox-LDL

Article Snippet: To analyze the effect of ox-LDL on HUVECs, the cells were treated with different concentrations (25, 50, 100, and 200 μg/ml) of ox-LDL for 24 h. In another experiment, HUVECs were pre-incubated with 100, 200, 400, and 800 pM of recombinant human Klotho (R&D Systems) protein for 1 h and subsequently treated with 50 μg/mlox-LDL for 24 h. These cells were then stained with 20 μl of 5 mg/ml MTT (Sigma-Aldrich) per well and incubated for 4 h at 37 °C.

Techniques: Microscopy, MTT Assay, Activity Assay, Incubation, Control

Journal: Lipids in Health and Disease

Article Title: Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways

doi: 10.1186/s12944-017-0447-0

Figure Lengend Snippet: Klotho inhibited ROS production induced by ox-LDL in HUVECs. HUVECs were pre-incubated with 200 pM of recombinant human Klotho for 1 h, then treated with ox-LDL (50 μg/mL) for another 24 h. Ox-LDL alone and Klotho alone were used as controls. a Images observed under an inverted fluorescence microscope. (Bar = 50 μm) ( b ) Output figure of the fluorescence intensity detected by flow cytometry. c Average fluorescence intensity: Mean = total area under the peak/the total number of cells. Data are shown as mean ± S.D. d Lipid peroxidation was assessed by measuring the MDA levels in HUVECs treated with ox-LDL and/or Klotho ( n = 3). Statistical differences are expressed as ## p < 0.01 vs. blank control; ** p < 0.01 vs. ox-LDL

Article Snippet: To analyze the effect of ox-LDL on HUVECs, the cells were treated with different concentrations (25, 50, 100, and 200 μg/ml) of ox-LDL for 24 h. In another experiment, HUVECs were pre-incubated with 100, 200, 400, and 800 pM of recombinant human Klotho (R&D Systems) protein for 1 h and subsequently treated with 50 μg/mlox-LDL for 24 h. These cells were then stained with 20 μl of 5 mg/ml MTT (Sigma-Aldrich) per well and incubated for 4 h at 37 °C.

Techniques: Incubation, Recombinant, Fluorescence, Microscopy, Flow Cytometry, Control

Journal: Lipids in Health and Disease

Article Title: Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways

doi: 10.1186/s12944-017-0447-0

Figure Lengend Snippet: SOD, Cu/Zn-SODandgp91 phox in ox-LDL and Klotho-treated HUVECs. Cellular treatment was the same as described in Fig. . a Intracellular SOD activity was determined by the hydroxylamine method. b , c and d Western blotanalysis of Cu/Zn-SOD and gp91 phoxexpression in pre-treated HUVECs. Densitometry of the probed bands from the western blot were analyzed by Image J2x. Values are means ± S.D. ( n = 3). Statistical differences are expressed as ## p < 0.01 vs. control; ** p < 0.01 vs. ox-LDL

Article Snippet: To analyze the effect of ox-LDL on HUVECs, the cells were treated with different concentrations (25, 50, 100, and 200 μg/ml) of ox-LDL for 24 h. In another experiment, HUVECs were pre-incubated with 100, 200, 400, and 800 pM of recombinant human Klotho (R&D Systems) protein for 1 h and subsequently treated with 50 μg/mlox-LDL for 24 h. These cells were then stained with 20 μl of 5 mg/ml MTT (Sigma-Aldrich) per well and incubated for 4 h at 37 °C.

Techniques: Activity Assay, Western Blot, Control

Journal: Lipids in Health and Disease

Article Title: Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways

doi: 10.1186/s12944-017-0447-0

Figure Lengend Snippet: Klotho regulated NO production in HUVECs. a NO production in pre-treated HUVECs. b , c , d , and e mRNA levels of iNOS, eNOS, PI3K, and Akt were measured by real time PCR. Values are means ± S.D. ( n = 3). Statistical differences are expressed as # p < 0.05 vs . control; * p < 0.05 vs . ox-LDL. ## p < 0.01 vs. control; ** p < 0.01 vs. ox-LDL

Article Snippet: To analyze the effect of ox-LDL on HUVECs, the cells were treated with different concentrations (25, 50, 100, and 200 μg/ml) of ox-LDL for 24 h. In another experiment, HUVECs were pre-incubated with 100, 200, 400, and 800 pM of recombinant human Klotho (R&D Systems) protein for 1 h and subsequently treated with 50 μg/mlox-LDL for 24 h. These cells were then stained with 20 μl of 5 mg/ml MTT (Sigma-Aldrich) per well and incubated for 4 h at 37 °C.

Techniques: Real-time Polymerase Chain Reaction, Control

Journal: Nature

Article Title: Structural basis for FGF hormone signalling.

doi: 10.1038/s41586-023-06155-9

Figure Lengend Snippet: Fig. 1 | HS promotes formation of 1:2:1:1 asymmetric FGF23–FGFR– αKlotho–HS quaternary complexes. Overall view of the cryo-EM reconstructions of FGF23–FGFR1c–αKlotho–HS (a), FGF23–FGFR3c–αKlotho– HS (b) and FGF23–FGFR4–αKlotho–HS (c) quaternary complexes displayed at threshold levels of 0.6, 0.45 and 0.5, respectively. The quaternary complex is shown in two different orientations related by a 180° rotation along the vertical axis. FGF23 is coloured in orange, αKlotho is shown in deep blue and

Article Snippet: For the FGFR1c+αKlothoTM co-expressing cell line, L6 cells stably expressing FGFR1cWT (resistant to G418) were infected with lentiviral particles encoding wild-type or mutated transmembrane αKlotho (αKlothoTM) and the co-expressing cells were selected using hygromycin (80 μg ml−1, no. HY-B0490, MedChemExpress).

Techniques: Cryo-EM Sample Prep

Journal: Nature

Article Title: Structural basis for FGF hormone signalling.

doi: 10.1038/s41586-023-06155-9

Figure Lengend Snippet: Fig. 2 | HS promotes asymmetric 1:2:1:1 FGF2–FGFR–αKlotho–HS quaternary complex by simultaneously engaging HBS of FGF23, FGFR1cP and FGFR1cS. a, FGF23–FGFR1c–αKlotho–HS asymmetric quaternary complex displayed as a hybrid of cartoon (FGF23, FGFR and HS) and surface (αKlotho) in the same orientation as in Fig. 1a (left). b, Expanded view of the boxed region in panel a showing tripartite interaction of HS with FGF23, FGFRP and FGFRS. HS interacting residues are shown as sticks and labelled. Hydrogen bonds in this figure and subsequent figures are represented as black dashed lines. Throughout the figures, nitrogen, oxygen and sulfur atoms are coloured blue, red and yellow, respectively. All structural illustrations were made using Pymol (v.2.5.2). c, L6-FGFR1cWT, L6-FGFR1cΔHBS1, L6-FGFR1cΔHBS1 or L6-FGFR1cΔHBS1+2 were cotreated with 20 nM 1:1 mixture of FGF23WT + αKlotho or FGF23R48A/R140A (FGF23ΔHBS) + αKlotho (in the case of L6-FGFR1cWT only) or left untreated. Total cell lysates were immunoblotted with an anti-pY656/Y657–FGFR, anti-pY783-PLCγ1,

Article Snippet: For the FGFR1c+αKlothoTM co-expressing cell line, L6 cells stably expressing FGFR1cWT (resistant to G418) were infected with lentiviral particles encoding wild-type or mutated transmembrane αKlotho (αKlothoTM) and the co-expressing cells were selected using hygromycin (80 μg ml−1, no. HY-B0490, MedChemExpress).

Techniques:

Journal: Nature

Article Title: Structural basis for FGF hormone signalling.

doi: 10.1038/s41586-023-06155-9

Figure Lengend Snippet: Fig. 3 | Direct FGFRP–FGFRS contacts are required for receptor dimerization and activation. a, Representation of the FGF23–FGFR1c–αKlotho–HS complex structure as a mix of cartoon (FGF23 and FGFR) and surface (αKlotho). View is related to that in Fig. 1a (right) by 90° rotation along the vertical axis. Contact sites 1 and 2 of the FGFR1cP–FGFR1cS dimer interface are boxed in blue and black, respectively. Note that αKlotho does not directly participate in recruiting FGFR1S. Left, magnified view of site 1 involving D2 domain of FGFR1cP and D2, D2–D3 linker and D3 of FGFR1cS. Right, close-up view of site 2 between D3 domains of FGFR1cP and FGFR1cS. Side chains of the interacting residues are shown as sticks. Selected secondary structure elements are labelled. Hydrophobic contacts are highlighted as a semitransparent surface. A Cu2+ ion (orange sphere) is coordinated by analogous histidine residues from FGFR1cP

Article Snippet: For the FGFR1c+αKlothoTM co-expressing cell line, L6 cells stably expressing FGFR1cWT (resistant to G418) were infected with lentiviral particles encoding wild-type or mutated transmembrane αKlotho (αKlothoTM) and the co-expressing cells were selected using hygromycin (80 μg ml−1, no. HY-B0490, MedChemExpress).

Techniques: Activation Assay

Journal: Nature

Article Title: Structural basis for FGF hormone signalling.

doi: 10.1038/s41586-023-06155-9

Figure Lengend Snippet: Fig. 4 | Secondary contacts between FGF23 and FGFRS are essential for receptor dimerization and activation. a, Cartoon representation of the FGF23–FGFR–αKlotho–HS structure in the same view as in Fig. 1a (right). Orange and black boxes signify the two contact regions between FGF23 and FGFRS, namely, FGF23core:FGFR1cS D2 domain (left, orange box) and FGF23NT: FGFR1cS D3 domain (right). b, L6-FGFR1cWT cells were treated with increasing concentrations of FGF23WT, FGF23ΔNT or FGF23ΔSRBS and whole cell lysates were probed as in Fig. 2. Equal homogeneity/quantity of FGF23WT, FGF23ΔNT and FGF23ΔSRBS samples was verified by SDS–PAGE (Supplementary Fig. 2d). c, Loss in the abilities of FGF23ΔNT and FGF23ΔSRBS mutants to induce FGF23–FGFR– αKlotho–HS quaternary complex formation (that is, receptor dimerization)

Article Snippet: For the FGFR1c+αKlothoTM co-expressing cell line, L6 cells stably expressing FGFR1cWT (resistant to G418) were infected with lentiviral particles encoding wild-type or mutated transmembrane αKlotho (αKlothoTM) and the co-expressing cells were selected using hygromycin (80 μg ml−1, no. HY-B0490, MedChemExpress).

Techniques: Activation Assay, SDS Page

Journal: British Journal of Pharmacology

Article Title: Genetic fusion of human FGF21 to a synthetic polypeptide improves pharmacokinetics and pharmacodynamics in a mouse model of obesity

doi: 10.1111/bph.13499

Figure Lengend Snippet: The characterization of FGF21 and fusion proteins. The unmodified FGF21 and fusion proteins were assayed by (A) 12% SDS‐PAGE and (B) Western blotting with a rabbit anti‐human FGF21 antibody (lane 1: FGF21; lane 2: PsTag200‐FGF21; lane 3: PsTag400‐FGF21; lane 4: PsTag600‐FGF21). (C) CD spectra of unmodified FGF21 and fusion proteins. (D) IEF of FGF21 and PsTag600‐FGF21 (lane 1: PsTag600‐FGF21; lane 2: FGF21). (E) MALDI‐TOF mass spectrometry of PsTag600‐FGF21 (M+ and M2 + refer to the singly and doubly charged ionic species of PsTag600‐FGF21 respectively. (F) SEC‐HPLC in the presence of 150 mmol·L−1 sodium phosphate buffer (pH 7.0) resulted in a single peak with decreasing elution time for PsTag fusion proteins with increasing number of amino acid residues. (G) Binding affinities of FGF21 and PsTag fusion proteins to human β‐klotho were examined by direct binding elisa. (H) Cellular glucose uptake stimulated by native FGF21 and PsTag fused FGF21 in 3 T3‐L1 cells. n = 3. *P < 0.05 versus vehicle control. (I) 3T3‐L1 cells were treated with vehicle (lane 1), 10 nmol·L−1 FGF21 (lane 2), 10 nmol·L−1 PsTag200‐FGF21 (lane 3), 10 nmol·L−1 PsTag400‐FGF21 (lane 4) and 10 nmol·L−1 PsTag600‐FGF21 (lane 5) for 10 min. Phospho‐specific antibody was used to determine phosphorylation of ERK. (J) Pharmacokinetic plasma profile of native FGF21 and PsTag fusion proteins intravenously injected in C57BL/6 mice (n = 10 per group).

Article Snippet: Binding affinities of FGF21 and PsTag fusion proteins to human β‐klotho (58 890 KB, R&D) were examined by direct binding elisa .

Techniques: SDS Page, Western Blot, Mass Spectrometry, Binding Assay, Enzyme-linked Immunosorbent Assay, Injection

Journal: PLoS ONE

Article Title: microRNA-200c regulates KLOTHO expression in human kidney cells under oxidative stress

doi: 10.1371/journal.pone.0218468

Figure Lengend Snippet: (A) Protein expression of KLOTHO in HK-2 cells treated with 100 μM H 2 O 2 for 24 hours (hrs). Band intensities were analyzed and normalized against levels of α-TUBULIN (TUBA) using densitometry. (B) Expression of KLOTHO mRNA in HK-2 cells treated with 100 μM H 2 O 2 for the indicated times. (C) Luciferase activities in HK-2 cells transfected with pMirTarget Vector harboring human KLOTHO 3′-UTR with or without 100 μM H 2 O 2 treatment for 12 hrs. Luciferase activity was normalized against protein amount. Values represent individual measurements and the mean ± SD. Data were analyzed using the Mann-Whitney U -test or the Mann-Whitney U -test with Bonferroni correction. * P < 0.05, n = 6.

Article Snippet: A KLOTHO 3′-UTR reporter clone in pMirTarget (pMirTarget-KL3′-UTR) was obtained from OriGene (SC217236, Rockville, MD).

Techniques: Expressing, Luciferase, Transfection, Plasmid Preparation, Activity Assay, MANN-WHITNEY

Journal: PLoS ONE

Article Title: microRNA-200c regulates KLOTHO expression in human kidney cells under oxidative stress

doi: 10.1371/journal.pone.0218468

Figure Lengend Snippet: (A) Putative miR-200c binding sites (underlined sequence) in the KLOTHO 3′-UTR sequence predicted by an online algorithm ( www.microrna.org ). (B) Predicted target sites of miR-200c in the KLOTHO mRNA 3′-UTR. There are two possible binding sites. (C) q-PCR analysis of pri-miR-200c and miR-200c expression in HK-2 cells cultured with or without 100 μM H 2 O 2 at the indicated time points. U6 snRNA was used for normalization. Values represent individual measurements and the mean ± SD. Data were analyzed using the Mann-Whitney U -test with Bonferroni correction. * P < 0.05, n = 6.

Article Snippet: A KLOTHO 3′-UTR reporter clone in pMirTarget (pMirTarget-KL3′-UTR) was obtained from OriGene (SC217236, Rockville, MD).

Techniques: Binding Assay, Sequencing, Expressing, Cell Culture, MANN-WHITNEY

Journal: PLoS ONE

Article Title: microRNA-200c regulates KLOTHO expression in human kidney cells under oxidative stress

doi: 10.1371/journal.pone.0218468

Figure Lengend Snippet: (A) KLOTHO protein expression in HK-2 cells 24 hrs after transfection with 25 nM mimic control or miR-200c mimic. Cells were cultured for another 48 hrs without mimic control or miR-200c mimic before sampling. Band intensities were analyzed and normalized against TUBA using densitometry. (B) A KLOTHO 3′-UTR reporter plasmid in combination with 50 nM mimic control or miR-200c mimic was transfected into HK-2 cells for 4 hrs. After a medium change, HK-2 cells were cultured for another 12 hrs before sampling. Luciferase activity was normalized against protein amount. (C) HK-2 cells were transfected with 50 nM mimic control or miR-200c mimic for 4 hrs and cultured for another 24 hrs before harvesting total RNA. KLOTHO mRNA levels were evaluated by q-PCR. (D) Mutations were introduced into the 3′-UTR of KLOHO mRNA as indicated. (E) The effect of 100 nM miR-200c mimic on the reporter activity of wild type (WT, pMirTarget-KL3′-UTR-WT) and mutant (MUT, pMirTarget-KL3′-UTR-MUT) plasmids in HK-2 cells was measured by luciferase assay. (F) HK-2 cells were stained with anti-KLOTHO antibody and Alexa Fluor 488-labeled goat anti-rabbit IgG. KLOTHO protein was evaluated under fluorescence microscopy. Scale bar = 10 μm. Values represent individual measurements and the mean ± SD. Data were analyzed using the Mann-Whitney U -test or the Mann-Whitney U -test with Bonferroni correction. * P < 0.05, n = 6. n.s.; not significant.

Article Snippet: A KLOTHO 3′-UTR reporter clone in pMirTarget (pMirTarget-KL3′-UTR) was obtained from OriGene (SC217236, Rockville, MD).

Techniques: Expressing, Transfection, Cell Culture, Sampling, Plasmid Preparation, Luciferase, Activity Assay, Mutagenesis, Staining, Labeling, Fluorescence, Microscopy, MANN-WHITNEY

Journal: Diabetology & Metabolic Syndrome

Article Title: Klotho and SIRT1 changes from pre-diabetes to diabetes and pre-hypertension to hypertension

doi: 10.1186/s13098-021-00736-2

Figure Lengend Snippet: Box and whisker plot of serum Klotho levels among different groups. The boxes represent the 25th and 75th percentiles, and the central lines in the boxes represent the median values. Whiskers are presented as median ± 95% CI. CTL control; Pre-DM pre-diabetes; DM diabetes mellitus; Pre-HTN pre-hypertension; HTN hypertension. **P < 0.01 vs. CTL, $$ P < 0.01 vs. Pre-DM, ## P < 0.01 vs. Pre-HTN

Article Snippet: Serum levels of Klotho and SIRT1 were measured by ELISA method using Human Klotho ELISA kit (R&D Systems with coefficient of variation < 10%) and Human SIRT1 ELISA kit (Elab Science with coefficient of variation < 10%).

Techniques: Whisker Assay, Control

Journal: Diabetology & Metabolic Syndrome

Article Title: Klotho and SIRT1 changes from pre-diabetes to diabetes and pre-hypertension to hypertension

doi: 10.1186/s13098-021-00736-2

Figure Lengend Snippet: Correlations of serum levels of Klotho ( A , C , E ) or SIRT1 ( B , D , F ) with FBS over the normal/Pre-DM ( A , B ) or normal to DM range ( C – F ). C , D linear, and E , F quadratic versions. CTL control; Pre-DM pre-diabetes; DM diabetes mellitus; FBS fasting blood sugar

Article Snippet: Serum levels of Klotho and SIRT1 were measured by ELISA method using Human Klotho ELISA kit (R&D Systems with coefficient of variation < 10%) and Human SIRT1 ELISA kit (Elab Science with coefficient of variation < 10%).

Techniques: Control

Journal: Diabetology & Metabolic Syndrome

Article Title: Klotho and SIRT1 changes from pre-diabetes to diabetes and pre-hypertension to hypertension

doi: 10.1186/s13098-021-00736-2

Figure Lengend Snippet: Correlations of serum levels of Klotho ( A , C , E ) or SIRT1 ( B , D , F ) with MAP over the normal/Pre-HTN ( A , B ) or normal to HTN ( C – F ). C , D linear, and E , F quadratic versions. CTL control; Pre-HTN pre-hypertension; HTN hypertension; MAP mean arterial pressure

Article Snippet: Serum levels of Klotho and SIRT1 were measured by ELISA method using Human Klotho ELISA kit (R&D Systems with coefficient of variation < 10%) and Human SIRT1 ELISA kit (Elab Science with coefficient of variation < 10%).

Techniques: Control

Journal: Diabetology & Metabolic Syndrome

Article Title: Klotho and SIRT1 changes from pre-diabetes to diabetes and pre-hypertension to hypertension

doi: 10.1186/s13098-021-00736-2

Figure Lengend Snippet: Linear regression of serum Klotho or SIRT1 level, as dependent variables, with age, BMI, FBS, MAP in studied groups

Article Snippet: Serum levels of Klotho and SIRT1 were measured by ELISA method using Human Klotho ELISA kit (R&D Systems with coefficient of variation < 10%) and Human SIRT1 ELISA kit (Elab Science with coefficient of variation < 10%).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Klotho-derived peptide KP1 ameliorates SARS-CoV-2-associated acute kidney injury

doi: 10.3389/fphar.2023.1333389

Figure Lengend Snippet: Klotho deficiency is a common feature of mouse kidney with preexisting chronic kidney diseases or advanced age. ( A – E ) Representative Western blots show renal protein level of Klotho in mouse models of ischemia-reperfusion injury (IRI), unilateral ureteral obstruction (UUO), remnant kidney after 5/6 nephrectomy (5/6Nx), db/db with diabetic kidney disease (DKD) and advanced age (24 months old), respectively. ( F ) Quantitative data of Klotho protein levels in the kidney of various models as indicated are presented. ** p < 0.01 versus sham ( n = 6). ( G ) Representative micrographs of immunochemical staining show Klotho expression and localization in different models of mice as indicated. Arrows indicate positive staining. Scale bar, 50 µm.

Article Snippet: Plasmid DNA-lipid complexes were added to the cells and incubated for 4–6 h. After transfection, cells were treated with KP1 (10 μg/mL) or recombinant human Klotho (100 ng/mL) (#5334-KL; R&D Systems) for 48 h. Cells were then collected and subjected to Western blot analyses or TUNEL staining, respectively.

Techniques: Western Blot, Staining, Expressing

Journal: Frontiers in Pharmacology

Article Title: Klotho-derived peptide KP1 ameliorates SARS-CoV-2-associated acute kidney injury

doi: 10.3389/fphar.2023.1333389

Figure Lengend Snippet: Klotho-deficiency sensitizes mice to SARS-CoV-2 N protein-triggered tubular injury and apoptosis. (A) Experimental design. The blue arrow indicates the timing of injecting pcDNA3 empty vector or pSARS-CoV-2 N Protein (N) plasmid into normal (WT) or Klotho-deficient ( KL/KL ) mice, respectively. (B, C) Graphic presentation shows serum creatinine (SCr) and blood urea nitrogen (BUN) levels in different groups. ns, not significant. (D–H) Representative Western blot (D) and quantitative data show renal protein levels of N Protein (F) , KIM-1 (G) and NGAL (H) in different groups. * p < 0.05, ** p < 0.01, *** p < 0.001 ( n = 6). ( I – L ) Representative Western blot (I) and quantitative data show renal protein levels of cleaved PARP (J) , p53 (K) and FADD ( L ). * p < 0.05, ** p < 0.01, *** p < 0.001 ( n = 6). ( M ) Representative micrographs of immunochemical staining show renal expression of N protein and P53 are presented. Arrows indicate positive staining. Scale bar, 50 µm. ( N ) Quantitative data of N Protein and p53. ** p < 0.01, *** p < 0.001 ( n = 6).

Article Snippet: Plasmid DNA-lipid complexes were added to the cells and incubated for 4–6 h. After transfection, cells were treated with KP1 (10 μg/mL) or recombinant human Klotho (100 ng/mL) (#5334-KL; R&D Systems) for 48 h. Cells were then collected and subjected to Western blot analyses or TUNEL staining, respectively.

Techniques: Plasmid Preparation, Western Blot, Staining, Expressing

Journal: Frontiers in Pharmacology

Article Title: Klotho-derived peptide KP1 ameliorates SARS-CoV-2-associated acute kidney injury

doi: 10.3389/fphar.2023.1333389

Figure Lengend Snippet: KP1 ameliorates tubular cell injury and apoptosis induced by SARS-CoV-2 N protein in vitro . (A–C) Representative Western blot (A) and quantitative data show the expression of N Protein (B) and KIM-1 (C) . Cells were treated with KP1 or recombinant human Klotho for 48 h after transfecting SARS-CoV-2 N Protein plasmid. ** p < 0.01, *** p < 0.001 ( n = 6). (D–G) Representative Western blot (D) and quantitative data show the expression of PARP (E) , p53 (F) and FADD (G) . * p < 0.05, ** p < 0.01, *** p < 0.001 ( n = 6). (H, I) Representative TUNEL staining (H) and quantitative data (I) show that SARS-CoV-2 N protein induced HK-2 cell apoptosis, while KP1 or Klotho (KL) protected HK-2 cells from apoptosis. ** p < 0.01 ( n = 6). Arrow indicates apoptotic cell.

Article Snippet: Plasmid DNA-lipid complexes were added to the cells and incubated for 4–6 h. After transfection, cells were treated with KP1 (10 μg/mL) or recombinant human Klotho (100 ng/mL) (#5334-KL; R&D Systems) for 48 h. Cells were then collected and subjected to Western blot analyses or TUNEL staining, respectively.

Techniques: In Vitro, Western Blot, Expressing, Recombinant, Plasmid Preparation, TUNEL Assay, Staining

Journal: Frontiers in Pharmacology

Article Title: Klotho-derived peptide KP1 ameliorates SARS-CoV-2-associated acute kidney injury

doi: 10.3389/fphar.2023.1333389

Figure Lengend Snippet: KP1 ameliorates acute kidney injury induced by SARS-CoV-2 N protein and ischemia-reperfusion injury in vivo . (A) Experimental design. The blue arrow indicates the timing of injecting pcDNA3 empty vector or pSARS-CoV-2 N Protein plasmid (N). The green arrowheads indicate the timing of injecting KP1 at the concentration of 1 mg/day/kg. The black arrow indicates the timing of IRI. (B, C) Graphic presentations show SCr and BUN levels in different groups as indicated. * p < 0.05, ** p < 0.01, *** p < 0.001 ( n = 6). (D–H) Representative Western blot (D) and quantitative data show renal protein levels of Klotho (E) , N protein (F) , KIM-1 (G) and NGAL (H) . * p < 0.05, ** p < 0.01, *** p < 0.001 ( n = 6). (I, J) Representative micrographs of Periodic acid-Schiff (PAS) staining and immunochemical staining for N protein and KIM-1 are presented. Arrows indicate positive staining. Scale bar, 50 µm. Semi-quantification data are presented in Panel (J) . * p < 0.05, ** p < 0.01, *** p < 0.001 ( n = 6).

Article Snippet: Plasmid DNA-lipid complexes were added to the cells and incubated for 4–6 h. After transfection, cells were treated with KP1 (10 μg/mL) or recombinant human Klotho (100 ng/mL) (#5334-KL; R&D Systems) for 48 h. Cells were then collected and subjected to Western blot analyses or TUNEL staining, respectively.

Techniques: In Vivo, Plasmid Preparation, Concentration Assay, Western Blot, Staining

Journal: Biogerontology

Article Title: Evaluation of potential aging biomarkers in healthy individuals: telomerase, AGEs, GDF11/15, sirtuin 1, NAD+, NLRP3, DNA/RNA damage, and klotho

doi: 10.1007/s10522-023-10054-x

Figure Lengend Snippet: Difference in the levels of aging biomarkers by age

Article Snippet: Klotho: using Human Klotho ELISA Kit (Cusabio, Cloud-Clone Corp, Katy, Texas, USA); samples were not diluted.

Techniques:

Journal: Biogerontology

Article Title: Evaluation of potential aging biomarkers in healthy individuals: telomerase, AGEs, GDF11/15, sirtuin 1, NAD+, NLRP3, DNA/RNA damage, and klotho

doi: 10.1007/s10522-023-10054-x

Figure Lengend Snippet: Descriptive data and p-values testing difference between male and female of aging biomarkers

Article Snippet: Klotho: using Human Klotho ELISA Kit (Cusabio, Cloud-Clone Corp, Katy, Texas, USA); samples were not diluted.

Techniques:

Journal: Biogerontology

Article Title: Evaluation of potential aging biomarkers in healthy individuals: telomerase, AGEs, GDF11/15, sirtuin 1, NAD+, NLRP3, DNA/RNA damage, and klotho

doi: 10.1007/s10522-023-10054-x

Figure Lengend Snippet: Correlations between parameters

Article Snippet: Klotho: using Human Klotho ELISA Kit (Cusabio, Cloud-Clone Corp, Katy, Texas, USA); samples were not diluted.

Techniques: