Journal: EBioMedicine

Article Title: Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/βKlotho Complex

doi: 10.1016/j.ebiom.2015.05.028

Figure Lengend Snippet: Selective activation of FGFR1c/KLB complex by bFKB1 (A) A proposed model for FGFR1c–KLB–bispecific Ab (bFKB1) complex formation for signal activation. D1, D2 and D3 denote each of the Immunoglobulin-like domains in FGFR1 extra cellular domain (ECD). KD: kinase domain. (B) A predicted model for FGFR1c–KLB–FGF21 complex formation for signal activation. (C) GAL–ELK1 luciferase assay of human FGF21 and bFKB1 activity using FGFR1 –KO HEK293T cells generated by CRISPR/Cas9-mediated genome editing. Cells were transfected to express indicated receptors. h: human, c: cynomolgus monkey, and m: murine. Mean relative luciferase unit (RLU) ± SEM is shown. (D) GAL–ELK1 luciferase assay in L6 cells. Cells were co-transfected to express indicated receptors. Transfected cells were incubated with various concentrations of bFKB1, hFGF21, hFGF19 or R1MAb1. Mean fold change ± SEM in RLU is shown. (E) Western blot analysis of primary human adipocytes treated with indicated protein (hFGF21 (100 nM) or IgG (33 nM)) for 1 h. Each treatment was performed in duplicate. (F) Expression of UCP1 mRNA in primary human adipocytes treated with indicated protein at 30 nM for 48 h. N = 3. p < 0.005 (**). Data are presented as mean fold over vehicle control ± SEM.

Article Snippet: Antibodies used for western blot analysis were from Cell Signaling Technology: pFRS2a (T196) (#3864), pMEK1/2 (S217/221) (#9154), MEK (#9126), pERK1/2 (T202/204) (#4370), ERK1/2 (#4695), HSP90 (#4874), β-Actin (#5125), from abcam: UCP1 (ab10983), or from R&D Systems: KLB (AF2619).

Techniques: Activation Assay, Luciferase, Activity Assay, Generated, CRISPR, Transfection, Incubation, Western Blot, Expressing, 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: Genetics Research

Article Title: Upregulation of Klotho Aggravates Insulin Resistance in Gestational Diabetes Mellitus Trophoblast Cells

doi: 10.1155/2022/1500768

Figure Lengend Snippet: Klotho affected insulin sensitivity. (a). RT-qPCR of klotho levels. (b) CCK-8 detection was used to detect cell viability. (c) The cells were subjected to a glucose uptake assay. (d) Western blot of GLUT4, INSR- α , INSR- β , IRS1, and IRS2 protein levels. ∗ p < 0.05 compared with the NC group. # p < 0.05 compared with the HG group.

Article Snippet: Anti-klotho antibody (28100-1-AP, proteintech), anti-Insulin receptor-alpha (INSR- α ) antibody (ab203037, Abcam), Insulin receptor-beta (INSR- β ) antibody (ab69508, Abcam), anti-Glucose transporter type 4 (GLUT4) antibody (66846-1-Ig, proteintech), anti-IRS1 antibody (17509-1-AP, proteintech), anti-IRS2 antibody (20702-1-AP, proteintech), anti-IGF-1 antibody (28530-1-AP, proteintech), anti-p-IGF-1R antibody (ab5681, Abcam), anti-IGF-1R antibody (20254-1-AP, proteintech), anti-p-mTOR antibody (67778-1-Ig, proteintech) anti-mTOR (66888-1-Ig, proteintech), anti-PI3K (ab140307, Abcam), anti-p-PI3K antibody (ab182651, Abcam), anti-Akt antibody (10176-2-AP, proteintech), anti-p-Akt (66444-1-Ig, proteintech) antibody and anti- β -actin antibody (66009-1-Ig, proteintech) were a primary antibody.

Techniques: Quantitative RT-PCR, CCK-8 Assay, Western Blot

Journal: Hepatology Communications

Article Title: Profiling of cell‐free DNA methylation and histone signatures in pediatric NAFLD: A pilot study

doi: 10.1002/hep4.2082

Figure Lengend Snippet: Comparison of characteristics in children without (no NASH) versus children with NASH

Article Snippet: The patatin‐like phospholipase domain containing 3 (PNPLA3) rs738409, transmembrane 6 superfamily member 2 (TM6SF2) rs58542926, membrane bound O‐acyltransferase domain containing 7 (MBOAT7) rs641738, and klotho‐β (KLB) rs17618244 variants were genotyped by allelic discrimination using TaqMan 5′‐nuclease assays (Life Technologies).

Techniques: Comparison

Journal: The EMBO Journal

Article Title: FGF23 promotes renal calcium reabsorption through the TRPV5 channel

doi: 10.1002/embj.201284188

Figure Lengend Snippet: Models of the regulation of apical membrane TRPV5 in renal distal tubules by Klotho and FGF23. Model based on previous studies for the regulation of apical membrane TRPV5 by secreted Klotho. TRPV5 is necessary for apical entry of calcium, which is then transported through the cell bound to calbindin D9k and D28k, and extruded at the basolateral side via PMCA1 and NCX. Secreted Klotho is thought to specifically hydrolyze sugar residues from the glycan chains on TRPV5 which in turn stabilizes TRPV5 in the membrane through interaction of the sugar residues with extracellular galectin (Chang et al , ; Cha et al , ). The cellular secretion process of Klotho in this model is unclear. Adapted from Cha et al . Our proposed model of Fgf23-αKlotho signaling in renal distal tubular cells. Fgf23 binds to the basolateral FGFR1c-Klotho complex and activates ERK1/2 leading to SGK1 phosphorylation. SGK1 in turn activates WNK4, stimulating WNK4-TRPV5 complex formation, and increasing intracellular transport of fully glycosylated TRPV5 from the Golgi apparatus to the plasma membrane. PTH signaling activates membrane-anchored TRPV5 by protein kinase A (PKA)-mediated phosphorylation.

Article Snippet: Serum Klotho protein concentrations were determined using a mouse specific Klotho ELISA kit (Cusabio) according to the manufacturer's protocol.

Techniques: Membrane, Glycoproteomics, Phospho-proteomics, Clinical Proteomics

Journal: The EMBO Journal

Article Title: FGF23 promotes renal calcium reabsorption through the TRPV5 channel

doi: 10.1002/embj.201284188

Figure Lengend Snippet: Renal calcium reabsorption and TRPV5 plasma membrane abundance in Fgf23 and Klotho deficient mouse models. A–D Urinary excretion of calcium corrected for creatinine (UrCa/Crea) (A), Western blotting quantification of core (75 kDa) and complex (92 kDa) glycosylated TRPV5 protein expression in renal cortical total membrane fractions (B), and Western blot analysis of membrane-bound Klotho in renal total protein extracts (C) in 9-month-old male WT, VDR Δ/Δ , Kl −/− /VDR Δ/Δ , and Fgf23 −/− /VDR Δ/Δ on rescue diet. Antibody specificity and glycosylation of TRPV5 and Klotho was controlled by a glycosylation assay (pink staining) followed by Western blot analysis of TRPV5 and Klotho, respectively (D). Anti-Klotho antibodies detecting the membrane-bound (anti-cytoplasmic domain, upper panel) or the membrane-bound and ectodomain shed (anti KL2 domain, lower panel) forms of the protein were used. Protein extracts from lung and spleen, as well as kidney extracts from Kl −/− /VDR Δ/Δ mice served as negative controls for TRPV5 and Klotho protein expression, respectively. Data information: * P < 0.05 vs. WT, # P < 0.05 vs. VDR Δ/Δ mice. Only the 135 kDa transmembrane isoform of Klotho was quantified in (C). Data in (A–C) represent mean ± s.e.m. of 4–9 animals each. Frames in Western blot images shown in (B) and (D) indicate splicing events. Source data are available online for this figure.

Article Snippet: Serum Klotho protein concentrations were determined using a mouse specific Klotho ELISA kit (Cusabio) according to the manufacturer's protocol.

Techniques: Clinical Proteomics, Membrane, Western Blot, Expressing, Glycoproteomics, Staining

Journal: The EMBO Journal

Article Title: FGF23 promotes renal calcium reabsorption through the TRPV5 channel

doi: 10.1002/embj.201284188

Figure Lengend Snippet: Membrane-bound αKlotho and TRPV5 do not co-localize in renal distal tubule cells. Immunohistochemical co-staining with anti-transmembrane αKlotho (red or green) antibody, anti-TRPV5 (green), anti-β-actin (red), and DAPI (blue) of paraffin sections from kidneys of 9-month-old WT, VDR Δ/Δ , and Fgf23 −/− /VDR Δ/Δ mice on rescue diet. Right panels show H&E-stained paraffin sections for comparison of subcellular localization. Scale bar, 20 μm. Immuno-electron microscopic staining using anti-Klotho antibodies against transmembrane, and transmembrane and shed forms in kidneys of WT (left panels) and Klotho −/− (right panels) mice. Upper panels show the apical cell area, lower panels show the basolateral area with the basal labyrinth. Arrows mark the apical cell membrane, asterisks mark mitochondria, and the symbol # marks the nucleus. Scale bar, 500 nm.

Article Snippet: Serum Klotho protein concentrations were determined using a mouse specific Klotho ELISA kit (Cusabio) according to the manufacturer's protocol.

Techniques: Membrane, Immunohistochemical staining, Staining, Comparison

Journal: The EMBO Journal

Article Title: FGF23 promotes renal calcium reabsorption through the TRPV5 channel

doi: 10.1002/embj.201284188

Figure Lengend Snippet: FGF23 increases urinary calcium reabsorption, TRPV5 plasma membrane abundance and activity in the kidney in gain-of-function mouse models. A, B Urinary calcium excretion (A) and serum PTH (B) in 4-month-old WT mice injected i.p. with vehicle or a single dose of 10 μg rFGF23 per mouse at time 0. C Urinary calcium excretion in 4-month-old WT, VDR Δ/Δ , and Kl −/− /VDR Δ/Δ mice on rescue diet injected i.p. with vehicle or a single dose of 10 μg rFGF23 per mouse, 8 h post-injection. D Immunohistochemical co-staining of kidney paraffin sections with anti-transmembrane αKlotho (red) antibody, anti-TRPV5 (green) and DAPI (blue). Original magnification ×630. E Western blot analysis of membrane-bound Klotho in renal total protein extracts from 4-month-old WT, VDR Δ/Δ , and Kl −/− /VDR Δ/Δ mice treated with vehicle or rFGF23 (10 μg/mouse) 8 h before necropsy. F Complex glycosylated TRPV5 protein expression in isolated distal tubular segments treated for 2 h in vitro with rFGF23 alone or in combination with a specific FGFR inhibitor (iFGFR). G Quantification and original images of intracellular Ca 2+ levels in renal distal tubular cells in 300-μm-thick kidney slices of 3-month-old WT mice treated with vehicle or rFGF23 (10 μg/mouse) 8 h before necropsy. Images are overlays of fluorescent with phase contrast images. Kidney slices were stained with the calcium-sensitive dye Fluo-4. Ruthenium red (10 μM) was used as TRPV inhibitor. H Time-dependent changes in distal tubular fluorescence in Fluo-4-loaded, 300-μm-thick kidney slices of 3-month-old WT mice treated at time 0 with rFGF23 (100 ng/ml) or vehicle. After 120, 135, and 150 min, 1, 10, and 50 μM of the TRPV inhibitor ruthenium red (RR) was added, respectively. Data information: Data in (A–C) represent mean ± s.e.m. of 3–5 animals each. * P < 0.05 vs. vehicle-treated control in (A–C). Data in (E) represent mean ± s.e.m. of 3–5 animals each. In (G), * P < 0.05 vs. vehicle-treated, # P < 0.05 vs. rFGF23-treated WT mice. In (H), * P < 0.05 vs. vehicle, # P < 0.05 vs. fluorescence at 120 min in rFGF23-treated slices. Fluorescence intensity in (G) and (H) was quantified in 7–9 regions of interest per experimental group and time point from three independent experiments. Scale bar, 20 μm in (D) and (G). Source data are available online for this figure.

Article Snippet: Serum Klotho protein concentrations were determined using a mouse specific Klotho ELISA kit (Cusabio) according to the manufacturer's protocol.

Techniques: Clinical Proteomics, Membrane, Activity Assay, Injection, Immunohistochemical staining, Staining, Western Blot, Expressing, Isolation, In Vitro, Fluorescence, Control

Journal: The EMBO Journal

Article Title: FGF23 promotes renal calcium reabsorption through the TRPV5 channel

doi: 10.1002/embj.201284188

Figure Lengend Snippet: FGF23 increases apical plasma membrane abundance of TRPV5 and causes re-distribution of WNK4. Immunohistochemical co-staining with anti-WNK4 (red) antibody, anti-TRPV5 (green) and DAPI (blue) of kidney paraffin sections from 4-month-old WT, VDR Δ/Δ and Klotho −/− /VDR Δ/Δ mice treated with vehicle or rFGF23 (10 μg/mouse) 8 h before necropsy. Scale bar, 20 μm. Right panel shows quantification of distal tubular TRPV5 and WNK4 co-localization performed in four animals per group with 3–6 images per animal and 4–6 different regions of interest per image. Immuno-electron microscopic staining using anti-TRPV5 (left panels) and anti-WNK4 (right panels) antibodies in kidneys from 3 to 4-month-old WT mice treated with vehicle (Veh) or rFGF23 (10 μg/mouse) 8 h before necropsy. Lower panels show higher magnification of the apical cell area from representative sections. Scale bar, 500 nm. Quantification of the relative grey levels in the apical area of the distal tubular cells between nucleus and apical cell membrane in the anti-TRPV5-stained immuno-electron microscopic pictures performed in four animals per group with 4–5 images per animal and 4–6 different regions of interest per image. Data information: * P < 0.05 vs. vehicle-treated controls in (A) and (C).

Article Snippet: Serum Klotho protein concentrations were determined using a mouse specific Klotho ELISA kit (Cusabio) according to the manufacturer's protocol.

Techniques: Clinical Proteomics, Membrane, Immunohistochemical staining, Staining

Journal: The EMBO Journal

Article Title: FGF23 promotes renal calcium reabsorption through the TRPV5 channel

doi: 10.1002/embj.201284188

Figure Lengend Snippet: FGF23 increases TRPV5 protein abundance and channel activity in MDCK cells transfected with murine TRPV5, SGK1 and WNK4. Complex glycosylated TRPV5 protein expression in total protein homogenates of MDCK cells transiently transfected with murine TRPV5 (T), SGK1 (S) and WNK4 (W) constructs, alone or in combination, and treated for 12 h with vehicle, rFGF23 or recombinant Klotho (rKlotho) alone or in combination. Mock-transfected cells were used as a negative control (Co). Quantification and original images of intracellular Ca 2+ levels in MDCK cells transiently transfected with murine TRPV5, SGK1 and WNK4 constructs after treatment with vehicle, rFGF23 or recombinant Klotho (rKlotho) for 12 h. Ruthenium red (10 μM) was used as TRPV inhibitor. Time-dependent changes and original images of intracellular Ca 2+ levels in MDCK cells transiently transfected with murine TRPV5, SGK1 and WNK4 constructs, and treated at time 0 with rFGF23 (100 ng/ml) or vehicle in the presence or absence of 1.5 mM EGTA added to the culture medium. After 105 min, 1 μM of ruthenium red (RR) was added. MDCK cells were stained with the calcium-sensitive dye Fluo-4 in (B) and (C). Data information: Data in (A) represent mean ± s.e.m. of 6–9 samples each from three independent experiments. Data in (B) and (C) represent mean ± s.e.m. of 3–4 samples each from three independent experiments. Scale bar, 20 μm in (B), 7 μm in (C). * P < 0.05 vs. vehicle in (A) and (B), and vs. EGTA + rFGF23 in (C), # P < 0.05 vs. fluorescence at 105 min in (C). Source data are available online for this figure.

Article Snippet: Serum Klotho protein concentrations were determined using a mouse specific Klotho ELISA kit (Cusabio) according to the manufacturer's protocol.

Techniques: Quantitative Proteomics, Activity Assay, Transfection, Expressing, Construct, Recombinant, Negative Control, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Biological effects of dosing aerobic exercise and neuromuscular electrical stimulation in rats

doi: 10.1038/s41598-017-11260-7

Figure Lengend Snippet: Comparison of systemic factors using enzyme-linked immunosorbent assays (ELISA). Brain-derived nerve growth factor (BDNF) was upregulated after both AE and NMES, with the most dramatic change evident after a medium dose of NMES resulting in an almost 8-fold increase. Little change was evident in VEGF-A, with only the low AE condition showing a minor non-significant 6% increase. The low AE condition also increased IGF-1 by 33% and showed an AE dose-dependent decrease, with the medium condition being equivalent to control levels. NMES did not affect IGF-1 levels. In contrast, Klotho revealed a significant AE dose-dependent modulation of levels with a medium dose of AE doubling the amount of Klotho in the blood compared to controls. NMES also produced an increase of Klotho for the low and high condition, but not for the medium dose. (*p < 0.05; **p < 0.01).

Article Snippet: Brain-derived neurotrophic factor (BDNF, ERBDNF, Thermo scientific), Vascular endothelial growth factor-A (VEGF-A, RRV00, R&D), Insulin-like growth factor 1 (IGF-1, CSB-E04582r, CUSABIO) and Klotho (CSB-E14958r, CUSABIO) levels were determined by quantitative enzyme-linked immunosorbent assay (ELISA) kits.

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Derivative Assay, Control, Produced

Journal: Scientific Reports

Article Title: Biological effects of dosing aerobic exercise and neuromuscular electrical stimulation in rats

doi: 10.1038/s41598-017-11260-7

Figure Lengend Snippet: Correlations between biological variables and MCT.

Article Snippet: Brain-derived neurotrophic factor (BDNF, ERBDNF, Thermo scientific), Vascular endothelial growth factor-A (VEGF-A, RRV00, R&D), Insulin-like growth factor 1 (IGF-1, CSB-E04582r, CUSABIO) and Klotho (CSB-E14958r, CUSABIO) levels were determined by quantitative enzyme-linked immunosorbent assay (ELISA) kits.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Biological effects of dosing aerobic exercise and neuromuscular electrical stimulation in rats

doi: 10.1038/s41598-017-11260-7

Figure Lengend Snippet: Contour maps – systemic factors. Based on the factorial design that arrayed treatment interventions to a control (0 dose), low, medium and high dose, contour plots of the dependent variables can be drawn to illustrate how these variables interact across the experimental space. It is evident here that systemic BDNF levels were most dramatically increased using NMES, but due to the lack of increase in MCT performance there was little interaction between treatment dose and % change in speed. BDNF did also not reveal a dramatic dose-dependent interaction for AE. A similar lack of interaction was evident for VEGF-A, with some evidence of an increase in levels for a low AE condition that results in a 25–50% change in speed. A clearer interaction between dosing and MCT was evident for AE on IGF-1. A low to medium dose of therapy here produced higher levels of factor release, but this was associated with a poorer performance. Lower levels of IGF-1 were actually induced by the high AE condition, producing the most significant performance change (upper right corner). Minimal interaction was seen for NMES, considering that there was less of an effect on MCT, demonstrating that IGF-1 levels in this paradigm were almost homogenous throughout experimental space. In the AE condition, the contour plot for Klotho indicated that the medium dose produced the highest level of Klotho release, which was associated with the highest level of behavioral change. In contrast the plot for NMES did not reveal a clear interaction between these variables.

Article Snippet: Brain-derived neurotrophic factor (BDNF, ERBDNF, Thermo scientific), Vascular endothelial growth factor-A (VEGF-A, RRV00, R&D), Insulin-like growth factor 1 (IGF-1, CSB-E04582r, CUSABIO) and Klotho (CSB-E14958r, CUSABIO) levels were determined by quantitative enzyme-linked immunosorbent assay (ELISA) kits.

Techniques: Control, Produced

Journal: Scientific Reports

Article Title: Biological effects of dosing aerobic exercise and neuromuscular electrical stimulation in rats

doi: 10.1038/s41598-017-11260-7

Figure Lengend Snippet: Hippocampal gene expression. BDNF mRNA was upregulated after AE, but did not exhibit a dose-dependency. A significant 10% increase in hippocampal BDNF was evident after low NMES and showed a dose dependent decrease to the level of controls. VEGF-A was upregulated in all NMES conditions, but did not change after a medium and high dose of AE. A low dose actually decreased VEGF-A expression. IGF-1 was upregulated in all conditions by approx. 17%. Only the low AE condition produced a lower 5% upregulation. Klotho revealed an expression profile similar to IGF-1 (27% increase), but with a more marked increase in expression in the low AE condition (12%). (*p < 0.05; **p < 0.01).

Article Snippet: Brain-derived neurotrophic factor (BDNF, ERBDNF, Thermo scientific), Vascular endothelial growth factor-A (VEGF-A, RRV00, R&D), Insulin-like growth factor 1 (IGF-1, CSB-E04582r, CUSABIO) and Klotho (CSB-E14958r, CUSABIO) levels were determined by quantitative enzyme-linked immunosorbent assay (ELISA) kits.

Techniques: Gene Expression, Expressing, Produced

Journal: Scientific Reports

Article Title: Biological effects of dosing aerobic exercise and neuromuscular electrical stimulation in rats

doi: 10.1038/s41598-017-11260-7

Figure Lengend Snippet: Correlations ELISA and mRNA.

Article Snippet: Brain-derived neurotrophic factor (BDNF, ERBDNF, Thermo scientific), Vascular endothelial growth factor-A (VEGF-A, RRV00, R&D), Insulin-like growth factor 1 (IGF-1, CSB-E04582r, CUSABIO) and Klotho (CSB-E14958r, CUSABIO) levels were determined by quantitative enzyme-linked immunosorbent assay (ELISA) kits.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Biological effects of dosing aerobic exercise and neuromuscular electrical stimulation in rats

doi: 10.1038/s41598-017-11260-7

Figure Lengend Snippet: Contour maps – hippocampal gene expression. By accounting for the different levels of dosing for AE and NMES, contour maps were generated to expose interactions between variables in the experimental space. It was evident here that BDNF expression in the hippocampus was highest for the high AE and low NMES conditions. As there was no significant improvement in MCT performance from NMES, this produced a rather flat banding of expression levels. The highest BDNF levels were evident between no change to a 25% performance decrease. For AE, the highest levels were achieved by a high dose that resulted in the highest level of performance increase. VEGF-A revealed the highest increase in the NMES group with a low to a medium dosing leading to peak gene expression. This was associated with improvements in MCT. AE yielded a dose-dependent increase in IGF-1 signaling that was linked to improved performance with a diagonal pattern across the experimental space. A similar effect was evident for Klotho with lower levels being associated with poorer performance and higher levels indicating better performance. A medium dosing of AE is sufficient to achieve peak performance and high levels of Klotho. It was also highly upregulated in the NMES condition with only very high decreases in performance showing a low level of Klotho.

Article Snippet: Brain-derived neurotrophic factor (BDNF, ERBDNF, Thermo scientific), Vascular endothelial growth factor-A (VEGF-A, RRV00, R&D), Insulin-like growth factor 1 (IGF-1, CSB-E04582r, CUSABIO) and Klotho (CSB-E14958r, CUSABIO) levels were determined by quantitative enzyme-linked immunosorbent assay (ELISA) kits.

Techniques: Gene Expression, Generated, Expressing, Produced

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