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: 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