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: 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: OncoTargets and Therapy

Article Title: Klotho ameliorates sepsis-induced acute kidney injury but is irrelevant to autophagy

doi: 10.2147/ott.s156891

Figure Lengend Snippet: Figure 2 Effects of recombinant KL, Rap, and 3-MA on kidney function and histopathology in mice with CLP-induced AKI. Notes: (A) Effects of recombinant KL, Rap, and 3-MA on Scr and BUN levels in mice 24 hours after CLP. *P,0.05 vs CLP+media. (B) Images of H&E staining of kidney tissues 24 hours after CLP showing vacuolar degeneration and loss of brush borders (black arrows) in proximal tubular epithelial cells. Images at 400× magnification were acquired using a biological imaging microscope (BX53; Olympus Corporation, Tokyo, Japan). The mice received recombinant KL (0.01 mg/kg) 1 hour after CLP. Rap (1 mg/kg) or 3-MA (30 mg/kg) was administered by intraperitoneal injection 2 hours before CLP surgery. Abbreviations: Rap, rapamycin; 3-MA, 3-methyladenine; CLP, cecal ligation and puncture; AKI, acute kidney injury; Scr, serum creatinine; BUN, blood urea nitrogen; CMC, carboxymethyl cellulose.

Article Snippet: Recombinant human KL protein was also purchased from R&D Systems, Inc. (catalog number: 5334-KL).

Techniques: Recombinant, Histopathology, Staining, Imaging, Microscopy, Injection, Ligation

Journal: OncoTargets and Therapy

Article Title: Klotho ameliorates sepsis-induced acute kidney injury but is irrelevant to autophagy

doi: 10.2147/ott.s156891

Figure Lengend Snippet: Figure 3 Effects of recombinant KL, Rap, and 3-MA on autophagy in mice with CLP-induced AKI. Notes: (A) Western blot for LC3-II, LC3-I, and P62. *P,0.05 vs CLP+media. #P,0.05 vs sham control. (B) Images of electron microscopy showing engulfment of degraded cytoplasmic components by autophagosomes (red arrows). The sections were observed under a Hitachi H7500 electron microscope (Hitachi Ltd., Tokyo, Japan). Mice received recombinant KL (0.01 mg/kg) 1 hour after CLP. Rap (1 mg/kg) or 3-MA (30 mg/kg) was administered by intraperitoneal injection 2 hours before CLP surgery. Abbreviations: Rap, rapamycin; 3-MA, 3-methyladenine; CLP, cecal ligation and puncture; AKI, acute kidney injury; CMC, carboxymethyl cellulose.

Article Snippet: Recombinant human KL protein was also purchased from R&D Systems, Inc. (catalog number: 5334-KL).

Techniques: Recombinant, Western Blot, Control, Electron Microscopy, Microscopy, Injection, Ligation

Journal: OncoTargets and Therapy

Article Title: Klotho ameliorates sepsis-induced acute kidney injury but is irrelevant to autophagy

doi: 10.2147/ott.s156891

Figure Lengend Snippet: Figure 4 Effects of recombinant KL, Rap, and 3-MA on endogenous renal KL expression in mice with CLP-induced AKI. Notes: (A) Western blot for endogenous renal KL expression. *P,0.05 vs CLP+media. (B) Images of IHC for KL. (C) Quantification of IHC staining. *P,0.05 vs CLP+media. Mice received recombinant KL (0.01 mg/kg) 1 hour after CLP. Rap (1 mg/kg) or 3-MA (30 mg/kg) was administered by intraperitoneal injection 2 hours before CLP surgery. Mouse renal tissues were collected 24 hours after CLP. Sections were stained with anti-KL (1:100, Abcam, Cambirdge, UK) at 4°C overnight. Images at 200× magnification were acquired using a biological imaging microscope (BX53; Olympus Corporation, Tokyo, Japan). Abbreviations: Rap, rapamycin; 3-MA, 3-methyladenine; CLP, cecal ligation and puncture; AKI, acute kidney injury; CMC, carboxymethyl cellulose; IHC, immunohistochemistry.

Article Snippet: Recombinant human KL protein was also purchased from R&D Systems, Inc. (catalog number: 5334-KL).

Techniques: Recombinant, Expressing, Western Blot, Immunohistochemistry, Injection, Staining, Imaging, Microscopy, Ligation

Journal: OncoTargets and Therapy

Article Title: Klotho ameliorates sepsis-induced acute kidney injury but is irrelevant to autophagy

doi: 10.2147/ott.s156891

Figure Lengend Snippet: Figure 5 Effects of recombinant KL, Rap, and 3-MA on renal CASP3 expression in mice with CLP-induced AKI. Notes: (A) Images of CASP3 IHC. (B) Quantitative analysis of IHC staining. *P,0.05 vs CLP+media. Mice received recombinant KL (0.01 mg/kg) 1 hour after CLP. Rap (1 mg/kg) or 3-MA (30 mg/kg) was administered by intraperitoneal injection 2 hours before CLP surgery. Mouse renal tissues were collected 24 hours after CLP. Sections were stained with anti-CASP3 (1:100; Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight. Images at 200× magnification were acquired using a biological imaging microscope (BX53; Olympus Corporation, Tokyo, Japan). Abbreviations: Rap, rapamycin; 3-MA, 3-methyladenine; CLP, cecal ligation and puncture; AKI, acute kidney injury; CMC, carboxymethyl cellulose; IHC, immunohistochemistry.

Article Snippet: Recombinant human KL protein was also purchased from R&D Systems, Inc. (catalog number: 5334-KL).

Techniques: Recombinant, Expressing, Immunohistochemistry, Injection, Staining, Imaging, Microscopy, Ligation

Journal: OncoTargets and Therapy

Article Title: Klotho ameliorates sepsis-induced acute kidney injury but is irrelevant to autophagy

doi: 10.2147/ott.s156891

Figure Lengend Snippet: Figure 7 Effects of recombinant KL, Rap, and 3-MA on LPS-treated HK-2 cells. Notes: (A) Effects of recombinant KL, Rap, and 3-MA on the autophagy of LPS-treated HK-2 cells. *P,0.05 vs LPS+media. (B) Effects of recombinant KL, Rap, and 3-MA on endogenous KL expression in LPS-treated HK-2 cells. Recombinant KL (0.4 μg/mL) was added 1 hour after incubation with LPS (10 μg/mL). Rap (1 µM) or 3-MA (5 mM) was added 30 min before LPS treatment to activate or suppress autophagy. Rap was dissolved in DMSO. DMSO was used as a control. Both culture media and cells were collected. The cells were harvested 24 hours after LPS exposure. Abbreviations: Rap, rapamycin; 3-MA, 3-methyladenine; LPS, lipopolysaccharide.

Article Snippet: Recombinant human KL protein was also purchased from R&D Systems, Inc. (catalog number: 5334-KL).

Techniques: Recombinant, Expressing, Incubation, Control

Journal: OncoTargets and Therapy

Article Title: Klotho ameliorates sepsis-induced acute kidney injury but is irrelevant to autophagy

doi: 10.2147/ott.s156891

Figure Lengend Snippet: Figure 8 Effects of recombinant KL, Rap, and 3-MA on apoptosis of LPS-treated HK-2 cells. Notes: (A) Scatter plot of apoptosis analysis. (B) Quantitative analysis of apoptosis. *P,0.05 vs LPS+media. HK-2 cells were exposed to recombinant KL (0.4 μg/mL) 1 hour after incubation with LPS (10 μg/mL). Rap (1 µM) or 3-MA (5 mM) was added 30 min before LPS treatment. The cells were trypsinized 24 hours after exposure to LPS and incubated in 500 μL of binding buffer containing 5 μL of Annexin V-FITC and 5 μL of propidium iodide (BD Biosciences, San Jose, CA, USA) in the dark for 15 min. The cells were analyzed in a flow cytometer. Flow cytometry data were analyzed using FlowJo software (Tree Star, San Carlos, CA, USA). Abbreviations: Rap, rapamycin; 3-MA, 3-methyladenine; LPS, lipopolysaccharide.

Article Snippet: Recombinant human KL protein was also purchased from R&D Systems, Inc. (catalog number: 5334-KL).

Techniques: Recombinant, Incubation, Binding Assay, Flow Cytometry, Software

Journal: OncoTargets and Therapy

Article Title: Klotho ameliorates sepsis-induced acute kidney injury but is irrelevant to autophagy

doi: 10.2147/ott.s156891

Figure Lengend Snippet: Figure 9 Autophagy-related signal in HK-2 cells following different treatments. Notes: Following treatment with recombinant KL (0.4 μg/mL), Rap (1 µM) and 3-MA (5 mM), the LC3-II fluorescence signal varied among the different groups. HK-2 cells cultured on coverslips were incubated with anti-LC3B (1:400) overnight at 4°C, incubated with donkey anti-rabbit IgG-conjugated FITC secondary antibody (1:200) for 1 hour at room temperature and viewed under a fluorescence microscope. Abbreviations: Rap, rapamycin; 3-MA, 3-methyladenine; LPS, lipopolysaccharide.

Article Snippet: Recombinant human KL protein was also purchased from R&D Systems, Inc. (catalog number: 5334-KL).

Techniques: Recombinant, Fluorescence, Cell Culture, Incubation, Microscopy

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: 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: FGF19 promotes the elongation of mitochondrial morphology by up-regulating the expression of mitochondrial fusion proteins. a RNA sequencing showing the change of mitochondrial metabolism-related genes 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). FPKM, Fragments per kilobase of exon model per million mapped fragments. b Representative western blotting showing the expression changes of Opa1, Mfn1 and Mfn2 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 Opa1, Mfn1 and Mfn2 by western blotting in b was performed to confirm these protein changes (n = 3). d Representative TEM images showing the changes of mitochondrial network’s morphology 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). Cyan arrows indicated the elongation of mitochondrial morphology. Schematic diagram illustrated that elongation was correlated with mitochondrial fusion. e Measurements of mitochondrial network’s morphology in d by Image J. Quantitative analyses of mitochondrial network’s morphology were based on three independent experiments (n = 3). 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: Expressing, RNA Sequencing, Western Blot, 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: FGF19 increases the mitochondrial biogenesis by up-regulating the expression of AMPKα signalling related proteins in chondrocytes. a RNA sequencing showing the change of FGFRs genes 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 q-PCR showing the gene changes of FGFR4 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). c Representative western blotting showing the expression change of FGFR4 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). d Representative western blotting showing the expression changes of AMPKα, p-AMPKα, PGC-1α and SIRT1 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). e Quantifications of AMPKα, p-AMPKα, PGC-1α and SIRT1 by western blotting in ( d ). f Representative immunofluorescent staining showing the change in the distribution of p-AMPKα 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-AMPKα; Green, F-actin; Blue, nucleus. g Quantification of fluorescence intensity of p-AMPKα in chondrocytes induced by FGF19 at 200 ng/ml in the presence of KLB (200 ng/ml). The data were based on at least eight cells from three independent experiments. h Representative immunofluorescent staining showing the change in the distribution of PGC-1α 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, PGC-1α; Green, F-actin; Blue, nucleus. i Quantification of fluorescence intensity of PGC-1α in chondrocytes induced by FGF19 at 200 ng/ml in the presence of KLB (200 ng/ml). The data were based on at least eight cells from three independent experiments. The data in g and i were shown as box (from 25, 50 to 75%) and whisker (minimum to maximum values) plots. The significant difference analysis in b , e , g and i 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: Expressing, RNA Sequencing, 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: 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: 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: 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: Inhibition of p38 decreases the expressions of mitochondrial fusion proteins induced by FGF19 in chondrocytes. a Representative western blotting showing the expression change of Opa1, Mfn1 and Mfn2 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 Opa1, Mfn1 and Mfn2 by western blotting in ( a ). c Representative immunofluorescent staining showing the change in the expression and distribution of Opa1 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, Opa1; Green, F-actin; Blue, nucleus. d Representative immunofluorescent staining showing the change in the distribution of Mfn2 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, Mfn2; Green, F-actin; Blue, nucleus. e Quantification of fluorescence intensity of Opa1 and Mfn2 in chondrocytes induced by SB203580 (10 µM) in the presence of FGF19 (200 ng/ml) and KLB (200 ng/ml). The data were based on at least eight cells from three independent experiments. f Representative immunofluorescent staining showing the changes of morphology mitochondrial network in living chondrocytes induced by SB203580 (10 µM) in the presence of FGF19 (200 ng/ml) and KLB (200 ng/ml) for 72 h. Image J shows the change of mitochondrial network morphology analysis in cyan boxes. The images were chosen based on three independent experiments (n = 3). Red, mitochondrial network; Blue, nucleus. g Quantification of mitochondrial number (per cell) and mitochondrial elongated number (per cell) in chondrocytes induced by SB203580 (10 µM) in the presence of FGF19 (200 ng/ml) by Image J. Quantitative analyses were based on three independent experiments (n = 3). The data in e and g were shown as box (from 25, 50 to 75%) and whisker (minimum to maximum values) plots. The significant difference analysis in b , e and g were 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, Western Blot, Expressing, Staining, Fluorescence, Whisker Assay

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Moderate aerobic exercise training decreases middle-aged induced pathologic cardiac hypertrophy by improving Klotho expression, MAPK signaling pathway, and oxidative stress status in Wistar rats

doi:

Figure Lengend Snippet: Serum levels of Klotho protein in young, middle-aged model, 4 weeks trained middle aged, and 8 weeks trained middle aged rats. Moderate exercise significantly increased Klotho in 8 weeks trained middle aged rats. ** P< 0.05 compared to young rats, ## P< 0.05 compared to middle-aged rats

Article Snippet: Finally, in the eight weeks, they reached a speed of 16 m/min, the slope of 0%, and distance traveled of 830 meters during 54 min ( ) ( Enzyme-linked immunosorbent assay (ELISA) The detection of Klotho protein in serum was performed using the corresponding ELISA kits (OKEH01598; Aviva Systems Biology Co, Ltd, CA, USA) based on standard sandwich ELISA technology.

Techniques: