recombinant fgf21 protein  (R&D Systems)

Journal: Journal of Inflammation Research

Article Title: FGF21 Exacerbates Obesity-Induced Airway Hyperresponsiveness and FGFR1-Dependent Mast Cell Activation in Mice

doi: 10.2147/JIR.S570000

Figure Lengend Snippet: FGF21 is upregulated in serum and BALF of obese mice, and positively correlated with airway resistance. Mice were fed either a standard chow diet (lean) or a high-fat diet (obese) for 16 weeks prior to analysis. ( A ) Measurements of body weight from the lean and obese group mice. ( B ) Representative pictures of lean and obese mice. ( C ) Levels of serum FGF21 in lean and obese mice were measured by ELISA (n=10). ( D ) Levels of BALF FGF21 in lean and obese mice were measured by ELISA (n=10). ( E ) DIO mice exhibit pronounced AHR. Results showed the changes in specific airway resistance (sRaw) as a measure of AHR. ( F ) Pearson’s correlation tests results represent FGF21 level in serum of obese mice was positively correlated with sRaw (C methacholine :50.0 mg/mL). ( G ) Pearson’s correlation tests results represent FGF21 level in BALF of obese mice was positively correlated with sRaw (C methacholine :50.0 mg/mL). Data are mean ± SEM, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant mouse FGF21 was purchased from MedChemExpress (HY- P72651 ).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation Research

Article Title: FGF21 Exacerbates Obesity-Induced Airway Hyperresponsiveness and FGFR1-Dependent Mast Cell Activation in Mice

doi: 10.2147/JIR.S570000

Figure Lengend Snippet: FGF21 level is increased in serum from obese patients with asthma and positively correlated with reduced pulmonary function. ( A ) Serum FGF21 levels in lean patients with asthma and obese patients with asthma were measured by ELISA (n=20-23). Data are mean ± SEM, * p < 0.05, *** p < 0.001 ( B ) Multivariable linear regression results represent serum FGF21 level was negatively correlated with FEV1%. ( C ) Multivariable linear regression results represent serum FGF21 level was negatively correlated with FEV1/FVC%, n=43.

Article Snippet: Recombinant mouse FGF21 was purchased from MedChemExpress (HY- P72651 ).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation Research

Article Title: FGF21 Exacerbates Obesity-Induced Airway Hyperresponsiveness and FGFR1-Dependent Mast Cell Activation in Mice

doi: 10.2147/JIR.S570000

Figure Lengend Snippet: Recombinant FGF21 aggravates AHR in obese mice, while Anti-FGF21 ameliorates obesity-induced AHR and inhibits mast cell infiltration. ( A ) The schematic diagram for recombinant FGF21 treatment in DIO mice. ( B ) Changes of airway resistance in DIO mice treated with recombinant FGF21. ( C ) Representative immunohistochemical images (× 400) for lung tissue sections staining of chymase. ( D ) Quantitative analysis of chymase staining. ( E ) The schematic diagram for Anti-FGF21 treatment in lean mice or DIO mice. ( F ) Changes of lung resistance (R L ) in DIO mice treated with the Anti-FGF21. ( G ) Representative immunohistochemical images (× 400) for lung tissue sections staining of chymase. ( H ) Quantitative analysis of chymase staining. n=5. Data are mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001. IgG: the normal IgG control antibody.

Article Snippet: Recombinant mouse FGF21 was purchased from MedChemExpress (HY- P72651 ).

Techniques: Recombinant, Immunohistochemical staining, Staining, Control

Journal: Journal of Inflammation Research

Article Title: FGF21 Exacerbates Obesity-Induced Airway Hyperresponsiveness and FGFR1-Dependent Mast Cell Activation in Mice

doi: 10.2147/JIR.S570000

Figure Lengend Snippet: FGF21 facilitates mast cell activation through up-regulating cholesterol biosynthesis. ( A ) Release of β-hexosaminidase from LAD2 and P815 cells pre-activated with compound 48/80 following 24 h treatment with recombinant FGF21 (100–400 ng/mL). ( B ) Release of histamine from LAD2 and P815 cells pre-activated with compound 48/80 following 24 h treatment with recombinant FGF21 (100–400 ng/mL). ( C ) Measurement of cellular calcium concentration in mast cells pre-activated with compound 48/80 using fluorescent probe Fluo-4 AM following 24 h treatment with recombinant FGF21 (200 ng/mL). ( D ) Quantitative analysis of fluorescence intensity of Fluo-4. ( E ) Filipin III staining of cholesterol in mast cells pre-activated with compound 48/80 following 24 h treatment with recombinant FGF21 (200 ng/mL). ( F ) Quantitative analysis of fluorescence intensity of Filipin III. ( G ) Expression levels of cholesterol biosynthesis genes in LAD2 cells. ( H ) Expression level of SREBF1 . ( I ) Quantitative analysis of fluorescence intensity of Filipin III in siRNA pretreated cells. ( J ) Representative images of Filipin III staining in siRNA-NC or siRNA- SREBF1 pretreated cells. ( K ) Release rate of β-hexosaminidase from LAD2 cells treated with siRNA and FGF21. ( L ) Measurement of cellular calcium concentration in LAD2 cells using fluorescent probe Fluo-4 AM. ( M ) Quantitative analysis of fluorescence intensity of Fluo-4 in LAD2 cells treated with siRNA and FGF21. n=3. Data are mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: Recombinant mouse FGF21 was purchased from MedChemExpress (HY- P72651 ).

Techniques: Activation Assay, Recombinant, Concentration Assay, Fluorescence, Staining, Expressing

Journal: Journal of Inflammation Research

Article Title: FGF21 Exacerbates Obesity-Induced Airway Hyperresponsiveness and FGFR1-Dependent Mast Cell Activation in Mice

doi: 10.2147/JIR.S570000

Figure Lengend Snippet: FGF21 promoted cholesterol synthesis and mast cell activation in a FGFR1-dependent manner. ( A ) Expression of FGFR1, FGFR2 and FGFR3 in lung tissues. (Data from Human Protein Atlas, http://www.proteinatlas.org/ ). ( B ) The mRNA expression of FGFR1, FGFR2 , and FGFR3 in LAD2 cells. Relative expression levels were normalized to GAPDH . ( C ) Release rate of β-hexosaminidase from LAD2 cells treated with FGFR1 inhibitor PD173074 . ( D ) Measurement of cellular calcium concentration in PD173074 treated LAD2 cells using fluorescent probe Fluo-4 AM. ( E ) Quantitative analysis of fluorescence intensity of Fluo-4 in LAD2 cells. ( F ) Expression levels of cholesterol biosynthesis genes in PD173074 treated LAD2 cells. ( G ) Representative images of Filipin III staining in PD173074 treated LAD2 cells. ( H ) Quantitative analysis of fluorescence intensity of Filipin III in PD173074 treated cells. n=3. Data are mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant mouse FGF21 was purchased from MedChemExpress (HY- P72651 ).

Techniques: Activation Assay, Expressing, Concentration Assay, Fluorescence, Staining

Journal: Journal of Inflammation Research

Article Title: FGF21 Exacerbates Obesity-Induced Airway Hyperresponsiveness and FGFR1-Dependent Mast Cell Activation in Mice

doi: 10.2147/JIR.S570000

Figure Lengend Snippet: Schematic of the role of FGF21 in obesity induced AHR. FGF21 promotes AHR in obese mice through increasing cholesterol synthesis and facilitating mast cell activation in a FGFR1-dependent manner.

Article Snippet: Recombinant mouse FGF21 was purchased from MedChemExpress (HY- P72651 ).

Techniques: Activation Assay

Journal: Physiological Reports

Article Title: Identification of FGF21 ‐inducing rare sugars that reduces sugar appetite in male BL /6 mice

doi: 10.14814/phy2.70618

Figure Lengend Snippet: Identification of FGF21‐inducing rare sugars and their effects on blood glucose. (a–d) FGF21 level of mouse primary hepatocytes treated for 24 h with vehicle (distilled water‐negative control) or 25 mM D‐glucose, D‐fructose (positive control), or rare sugars (46 samples). n = 3 mice. (e) Plasma FGF21 levels over 24 h after gastric gavage of vehicle (distilled water), D‐glucose, D‐tagatose, D‐allulose, or D‐sorbitol at 5 g/kg in nine‐week‐old male WT mice with ad libitum access to normal chow (NC) diet. n = 4 mice per group. (f) Area under the curve (AUC) over 24 h of data in (e). n = 4 mice per group. (g) Blood glucose levels of the mice receiving the same dose in (e) of either vehicle (distilled water), D‐glucose, D‐tagatose, D‐allulose, or D‐sorbitol after 16 h fasting. n = 4 mice per group. Statistical analyses were done by one‐way ANOVA for (a–d, f) and repeated measures ANOVA for (e, g), followed by Tukey's HSD test. ** p < 0.01; **** p < 0.0001.

Article Snippet: Mouse FGF21 protein levels in each plasma sample were determined using a Mouse/Rat FGF‐21 Quantikine ELISA Kit (R&D Systems) according to the manufacturer's instructions.

Techniques: Negative Control, Positive Control, Clinical Proteomics

Journal: Physiological Reports

Article Title: Identification of FGF21 ‐inducing rare sugars that reduces sugar appetite in male BL /6 mice

doi: 10.14814/phy2.70618

Figure Lengend Snippet: FGF21‐inducing rare sugars activated oxytocin neurons of paraventricular nucleus of hypothalamus in BL/6 mice. (a–e) Immunostaining of mice brain after gastric gavage with either (a) vehicle (distilled water) or 5 g/kg body weight of D‐glucose (b), D‐tagatose (c), D‐allulose (d), or D‐sorbitol (e). Blue arrow ( ) indicates c‐Fos‐positive oxytocin neurons, shown by dark brown spot on the light brown neurons ( ). Magenta arrow ( ) indicates c‐Fos‐negative oxytocin neurons, shown by light brown anti‐oxytocin staining ( ). n = 4 mice per group. Scale bar represents 100 μ m. (f) Percentage of activated oxytocin neurons (c‐Fos positive oxytocin neurons) from the total of PVH oxytocin neurons in the mice receiving either vehicle (distilled water), D‐glucose, or FGF21‐inducing rare sugars in (a–e). Data are presented as box‐whiskers in (f), where the middle line represents median, bottom/ top edges represent 25th/75th percentile of the data, and whiskers represent maximum and minimum value of the data. Statistical analyses were done by one‐way ANOVA, followed by Dunnett's test.

Article Snippet: Mouse FGF21 protein levels in each plasma sample were determined using a Mouse/Rat FGF‐21 Quantikine ELISA Kit (R&D Systems) according to the manufacturer's instructions.

Techniques: Immunostaining, Staining

Journal: Physiological Reports

Article Title: Identification of FGF21 ‐inducing rare sugars that reduces sugar appetite in male BL /6 mice

doi: 10.14814/phy2.70618

Figure Lengend Snippet: Intragastric administration of FGF21‐inducing rare sugars reduced sucrose preference in BL/6 mice. (a–c) 50 mM sucrose intake in mice receiving 5 g/kg body weight of D‐allulose (a), D‐tagatose (b), or D‐sorbitol (c). (d–f) sucrose preference of the same mice in (a–c). n = 10 mice per group for (a, d) and n = 5 mice per group for (b, c, e, f). Data are presented as box‐whiskers, where the middle line represents the median, bottom/top edges represent the 25th/75th percentile of the data, and whiskers represent the maximum and minimum value of the data. Statistical analyses were done by Student's paired t ‐test between “pre” and “post” of each treatment.

Article Snippet: Mouse FGF21 protein levels in each plasma sample were determined using a Mouse/Rat FGF‐21 Quantikine ELISA Kit (R&D Systems) according to the manufacturer's instructions.

Techniques:

Journal: Physiological Reports

Article Title: Identification of FGF21 ‐inducing rare sugars that reduces sugar appetite in male BL /6 mice

doi: 10.14814/phy2.70618

Figure Lengend Snippet: Mixing FGF21‐inducing rare sugars into sucrose solution reduced solution intake and preference in BL/6 mice. (a–c) Intake of 100 mM (final concentration) sucrose solution that was mixed with either vehicle (distilled water), 600 mM (final concentration) of D‐glucose, or 600 mM (final concentration) of D‐allulose (a), D‐tagatose (b), or D‐sorbitol (c). (d–f) Sucrose preference of the same mice in (a–c). n = 6 mice per group. Data are presented as box‐whiskers, where the middle line represents the median, bottom/top edges represent the 25th/75th percentile of the data, and whiskers represent the maximum and minimum value of the data. Statistical analyses were done by repeated measures ANOVA, followed by Tukey's HSD test.

Article Snippet: Mouse FGF21 protein levels in each plasma sample were determined using a Mouse/Rat FGF‐21 Quantikine ELISA Kit (R&D Systems) according to the manufacturer's instructions.

Techniques: Concentration Assay

Journal: Neuropsychiatric Disease and Treatment

Article Title: Fibroblast Growth Factor 21 Protects Against Cerebral Ischemia/Reperfusion Injury by Inhibiting Oxidative Stress and Ferroptosis

doi: 10.2147/NDT.S504180

Figure Lengend Snippet: FGF21 reduces the cerebral infarction volume and decreases the number of apoptotic cells in mice with IRI. ( A ) TTC staining: Cerebral infarction. ( B ) TUNEL staining: Cellular apoptosis. Data represent Mean ± SD. (n=5). * P < 0.05, *** P < 0.001, ns, no significance. The red arrows: TUNEL positive cells (apoptotic cells).

Article Snippet: The methodology and FGF21 dosages used were based on prior studies of FGF21 in treating BBB injury and ventilator‐induced lung injury., Briefly, recombinant mouse FGF21 (1.5 mg/kg, P6101, Beyotime, Shanghai, China) was dissolved in PBS and injected intraperitoneally 15 min pre-reperfusion, 8 and 16 h post-reperfusion.

Techniques: Staining, TUNEL Assay

Journal: Neuropsychiatric Disease and Treatment

Article Title: Fibroblast Growth Factor 21 Protects Against Cerebral Ischemia/Reperfusion Injury by Inhibiting Oxidative Stress and Ferroptosis

doi: 10.2147/NDT.S504180

Figure Lengend Snippet: FGF21 attenuates pathological damage to the brain tissue and neuronal injury in CIR mice. ( A ) H&E staining: Pathological damage to the brain tissue. ( B ) Nissl staining: Neuronal survival. Scale bar: 500 and 50 μm (low- and high-magnification images). The red box: Acquisition area for the high-magnification image.

Article Snippet: The methodology and FGF21 dosages used were based on prior studies of FGF21 in treating BBB injury and ventilator‐induced lung injury., Briefly, recombinant mouse FGF21 (1.5 mg/kg, P6101, Beyotime, Shanghai, China) was dissolved in PBS and injected intraperitoneally 15 min pre-reperfusion, 8 and 16 h post-reperfusion.

Techniques: Staining

Journal: Neuropsychiatric Disease and Treatment

Article Title: Fibroblast Growth Factor 21 Protects Against Cerebral Ischemia/Reperfusion Injury by Inhibiting Oxidative Stress and Ferroptosis

doi: 10.2147/NDT.S504180

Figure Lengend Snippet: Inhibitory effect of FGF21 on oxidative stress (OxS) in mice post-IRI. ELISA: Levels of OxS products in the serum: ( A ) MDA, ( B ) MPO, ( C ) ROS, and ( D ) SOD. Data represent Mean ± SD. (n=5). * P < 0.05, ** P < 0.01, *** P < 0.001, ns, no significance.

Article Snippet: The methodology and FGF21 dosages used were based on prior studies of FGF21 in treating BBB injury and ventilator‐induced lung injury., Briefly, recombinant mouse FGF21 (1.5 mg/kg, P6101, Beyotime, Shanghai, China) was dissolved in PBS and injected intraperitoneally 15 min pre-reperfusion, 8 and 16 h post-reperfusion.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Neuropsychiatric Disease and Treatment

Article Title: Fibroblast Growth Factor 21 Protects Against Cerebral Ischemia/Reperfusion Injury by Inhibiting Oxidative Stress and Ferroptosis

doi: 10.2147/NDT.S504180

Figure Lengend Snippet: FGF21 inhibits ferroptosis in mouse brain tissue post-IRI. ELISA: ( A ) Concentration of ROS, ( B ) Fe² + , ( C ) GSSG, and ( D ) GSH. Western blotting: ( E ) bands of ferroptosis-related proteins, ( F ) expression levels of ferroptosis-promoting protein ACSL4 and ( G ) ferroptosis-inhibiting protein GPX4. Data represent Mean ± SD; (n = 5). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The methodology and FGF21 dosages used were based on prior studies of FGF21 in treating BBB injury and ventilator‐induced lung injury., Briefly, recombinant mouse FGF21 (1.5 mg/kg, P6101, Beyotime, Shanghai, China) was dissolved in PBS and injected intraperitoneally 15 min pre-reperfusion, 8 and 16 h post-reperfusion.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, Expressing

Journal: Neuropsychiatric Disease and Treatment

Article Title: Fibroblast Growth Factor 21 Protects Against Cerebral Ischemia/Reperfusion Injury by Inhibiting Oxidative Stress and Ferroptosis

doi: 10.2147/NDT.S504180

Figure Lengend Snippet: RNA sequencing of ischemic penumbra tissue screening for DEGs. Volcano plot: DEGs following ( A ) MCAO/R injury vs Sham group and ( B ) following FGF21 treatment vs MCAO/R group. Heatmap: Top 50 DEGs following ( C ) MCAO/R injury vs Sham group and ( D ) following FGF21 treatment vs MCAO/R group.

Article Snippet: The methodology and FGF21 dosages used were based on prior studies of FGF21 in treating BBB injury and ventilator‐induced lung injury., Briefly, recombinant mouse FGF21 (1.5 mg/kg, P6101, Beyotime, Shanghai, China) was dissolved in PBS and injected intraperitoneally 15 min pre-reperfusion, 8 and 16 h post-reperfusion.

Techniques: RNA Sequencing

Journal: Neuropsychiatric Disease and Treatment

Article Title: Fibroblast Growth Factor 21 Protects Against Cerebral Ischemia/Reperfusion Injury by Inhibiting Oxidative Stress and Ferroptosis

doi: 10.2147/NDT.S504180

Figure Lengend Snippet: GO, KEGG, and Reactome enrichment analyses of DEGs. Compared to Sham group: ( A ) Bar charts: GO analysis of DEGs after MCAO/R injury; ( B ) Bubble plot: KEGG analysis, highlighting the top 30 significantly enriched pathways after MCAO/R injury; ( C ) Bar charts: Reactome analysis after MCAO/R injury. Compared to the MCAO/R group: ( D ) Bar charts: GO analysis of DEGs after FGF21 treatment; ( E ) Bubble plot: KEGG analysis, emphasizing the top 30 significantly enriched pathways after FGF21 treatment; ( F ) Bar charts: Reactome analysis after FGF21 treatment.

Article Snippet: The methodology and FGF21 dosages used were based on prior studies of FGF21 in treating BBB injury and ventilator‐induced lung injury., Briefly, recombinant mouse FGF21 (1.5 mg/kg, P6101, Beyotime, Shanghai, China) was dissolved in PBS and injected intraperitoneally 15 min pre-reperfusion, 8 and 16 h post-reperfusion.

Techniques:

Journal: Neuropsychiatric Disease and Treatment

Article Title: Fibroblast Growth Factor 21 Protects Against Cerebral Ischemia/Reperfusion Injury by Inhibiting Oxidative Stress and Ferroptosis

doi: 10.2147/NDT.S504180

Figure Lengend Snippet: CYBB may be a key effector of FGF21, crucial for inhibiting ferroptosis. ( A ) Venn diagram: Overlap between DEGs and ferroptosis-related genes. ( B ) Comparison of significant differential expression rates for ferroptosis-related vs other genes. ( C ) RNA sequencing: Expression fold changes for the top four significantly altered ferroptosis-related genes. ( D ) qPCR: Relative expression levels for the top four ferroptosis-related genes with significant fold changes. ( E ) Western blotting: CYBB protein expression levels. ( F ) Immunofluorescence double-labeling staining: CYBB and the neuronal-specific marker NeuN. Data represent Mean ± SD; (n = 5). ** P < 0.01, *** P < 0.001. The red arrows: Double-labeled immunofluorescent positive cells.

Article Snippet: The methodology and FGF21 dosages used were based on prior studies of FGF21 in treating BBB injury and ventilator‐induced lung injury., Briefly, recombinant mouse FGF21 (1.5 mg/kg, P6101, Beyotime, Shanghai, China) was dissolved in PBS and injected intraperitoneally 15 min pre-reperfusion, 8 and 16 h post-reperfusion.

Techniques: Comparison, Quantitative Proteomics, RNA Sequencing, Expressing, Western Blot, Immunofluorescence, Labeling, Staining, Marker

Journal: Neuropsychiatric Disease and Treatment

Article Title: Fibroblast Growth Factor 21 Protects Against Cerebral Ischemia/Reperfusion Injury by Inhibiting Oxidative Stress and Ferroptosis

doi: 10.2147/NDT.S504180

Figure Lengend Snippet: Schematic representation of the mechanisms by which FGF21 influences CIRI. FGF21 protects CIRI by inhibiting OxS and ferroptosis; CYBB, a new key regulator, may mediate its anti-ferroptotic effects.

Article Snippet: The methodology and FGF21 dosages used were based on prior studies of FGF21 in treating BBB injury and ventilator‐induced lung injury., Briefly, recombinant mouse FGF21 (1.5 mg/kg, P6101, Beyotime, Shanghai, China) was dissolved in PBS and injected intraperitoneally 15 min pre-reperfusion, 8 and 16 h post-reperfusion.

Techniques:

Journal: bioRxiv

Article Title: The myokine FGF21 associates with enhanced survival in ALS and mitigates stress-induced cytotoxicity

doi: 10.1101/2024.09.11.611693

Figure Lengend Snippet: ( A ) FGF21 mRNA expression was analysed in normal ( n = 24) and ALS ( n = 36) muscle biopsy samples via RT-qPCR. ** P = 0.004, unpaired two-tailed t-test with Welch’s correction. ( B ) FGF21 mRNA levels were quantified in normal ( n = 7) and ALS ( n = 11) post-mortem muscle samples (left panel) and FGF21 protein levels ( n = 13 for normal samples; n = 18 for ALS samples; right panel). ** P = 0.003, **** P < 0.0001, two-tailed Mann Whitney test. (C) FGF21 mRNA levels were quantified in normal ( n = 14) and ALS ( n = 22) post-mortem spinal cord samples (left panel) and FGF21 protein levels ( n = 12 for normal samples; n = 18 for ALS samples; right panel). ** P = 0.00, two-tailed Mann Whitney test. (D) Comparison of spinal cord and muscle FGF21 protein levels for 18 ALS patients. A spearman correlation test was used for analysis. (E) FGF21 mRNA levels in the gastrocnemius muscle (left panel) and spinal cord (right panel) were quantified across different age groups (20 – 150 days; n = 4-5 per group) from SOD1 G93A mice and littermate controls. ** P < 0.01, *** P < 0.001, **** P < 0.0001, unpaired two-tailed t-test comparing WT to SOD1 G93A . For all graphs, error bars represent SD.

Article Snippet: Cells were treated with methionine-cystine (MetCys)-deprived media (Thermo Fisher Scientific) or 100 ng/ml mouse recombinant FGF21 (GenScript) or exposed to 100 μM H 2 0 2 (Sigma Aldrich) for 24 h. Additionally, some cells were transfected with pCDNA3.1 FGF21-Flag (GenScript), Flag-SOD1 G93A and Flag-SOD1WT plasmid constructs using Lipofectamine 2000 (Thermo Fisher Scientific) in Opti-MEM Reduced Serum Media (Thermo Fisher Scientific).

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: bioRxiv

Article Title: The myokine FGF21 associates with enhanced survival in ALS and mitigates stress-induced cytotoxicity

doi: 10.1101/2024.09.11.611693

Figure Lengend Snippet: (A) Tissue sections from two ALS patients and one normal control were immunostained with an anti-FGF21 antibody and counterstained with Hoechst and wheat germ agglutin (WGA). Intense immunoreactivity is observed in atrophic myofibers (asterisks) and in the endomysial space (arrows) in the ALS muscle sections. Scale bars, 100 µM in low power views and 50 µM in the enlarged views. (B) Mean fluorescence Intensity (MFI) analysis of FGF21 immunoreactivity was performed in 5 ALS patient biopsy samples and 5 normal controls. Atrophic (< 25 μM minimal Feret’s diameter) and non-atrophic myofibers were selected in the same section as shown in the micrograph (yellow outline). FGF21 MFI (per μM 2 ) was quantitated for 46 atrophic and non-atrophic myofibers and summarized in the graph (horizontal line represents the mean). **** P < 0.0001; two-tailed Mann Whitney test. Scale bar: 50 µM.

Article Snippet: Cells were treated with methionine-cystine (MetCys)-deprived media (Thermo Fisher Scientific) or 100 ng/ml mouse recombinant FGF21 (GenScript) or exposed to 100 μM H 2 0 2 (Sigma Aldrich) for 24 h. Additionally, some cells were transfected with pCDNA3.1 FGF21-Flag (GenScript), Flag-SOD1 G93A and Flag-SOD1WT plasmid constructs using Lipofectamine 2000 (Thermo Fisher Scientific) in Opti-MEM Reduced Serum Media (Thermo Fisher Scientific).

Techniques: Control, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: The myokine FGF21 associates with enhanced survival in ALS and mitigates stress-induced cytotoxicity

doi: 10.1101/2024.09.11.611693

Figure Lengend Snippet: (A) Plasma samples from age-matched normal controls ( n = 23) and ALS patients ( n = 28) and assayed by ELISA for FGF21. * P = 0.043, unpaired two-tailed t-test. (B) 16 ALS patients from a prior biomarker study were divided into slow ( n = 6), average ( n = 5), and fast ( n = 5) progressing groups based on the average in the study monthly decline in ALSFRS-R scores. (C) Plasma FGF21 levels were measured at baseline and 3 months and averaged. The normal control values were added for comparison. ** P = 0.003, *** P = 0.0003; one-way ANOVA followed by Tukey post hoc test. (D) Correlation between plasma FGF21 levels with monthly change in the ALSFRS-R scores (ΔFRS) for each subject. Spearman correlation test. (E) Kaplan–Meier survival curves for study patients whose FGF21 plasma levels were < 1.5-fold-change (FC) over the normal control group versus study patients with ≥ 1.5-FC in FGF21 levels. * P = 0.015; Log-rank (Mantel-Cox) test. (F) Comparison of body mass index (BMI) between the < 1.5 FC and ≥ 1.5-FC groups. * P = 0.037; unpaired two-tailed t-test. For all graphs, error bars represent SD.

Article Snippet: Cells were treated with methionine-cystine (MetCys)-deprived media (Thermo Fisher Scientific) or 100 ng/ml mouse recombinant FGF21 (GenScript) or exposed to 100 μM H 2 0 2 (Sigma Aldrich) for 24 h. Additionally, some cells were transfected with pCDNA3.1 FGF21-Flag (GenScript), Flag-SOD1 G93A and Flag-SOD1WT plasmid constructs using Lipofectamine 2000 (Thermo Fisher Scientific) in Opti-MEM Reduced Serum Media (Thermo Fisher Scientific).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Biomarker Discovery, Control, Comparison

Journal: bioRxiv

Article Title: The myokine FGF21 associates with enhanced survival in ALS and mitigates stress-induced cytotoxicity

doi: 10.1101/2024.09.11.611693

Figure Lengend Snippet: (A) FGF21 were measured in iPSC-derived motor neurons obtained from healthy controls and ALS patients carrying either C9orf72 or SOD1 mutations (Supplementary Table 1). * P = 0.012; two-tailed Mann Whitney test. (B) KLB mRNA levels were similarly measured in iPSC motor neurons. * P = 0.018; two-tailed Mann Whitney test. (C) NSC-34 motor neuron-like cells were transfected with FLAG-tagged WT and SOD1 G93A expression plasmids and lysates were assessed by western blot using the antibodies indicated. Bands were quantitated by densitometry and a ratio to the loading control, vinculin, was calculated (shown between the two blots). (D) KLB and (E) FGF21 mRNA levels were measured in the same lysates. * P = 0.018, *** P = 0.0002, unpaired two-tailed t-test. (F) FGF21 protein was measured in the CM of transfected NSC-34 cells. (G) FGF21 or (H) KLB mRNA levels were quantified from NSC-34 cells exposed to methionine-cystine (MetCys)-deprived media or treated with 100μM H 2 O 2 for 24 h. * P = 0.048, **** P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparisons test. Data points represent biological replicates and bars are the mean ± SD.

Article Snippet: Cells were treated with methionine-cystine (MetCys)-deprived media (Thermo Fisher Scientific) or 100 ng/ml mouse recombinant FGF21 (GenScript) or exposed to 100 μM H 2 0 2 (Sigma Aldrich) for 24 h. Additionally, some cells were transfected with pCDNA3.1 FGF21-Flag (GenScript), Flag-SOD1 G93A and Flag-SOD1WT plasmid constructs using Lipofectamine 2000 (Thermo Fisher Scientific) in Opti-MEM Reduced Serum Media (Thermo Fisher Scientific).

Techniques: Derivative Assay, Two Tailed Test, MANN-WHITNEY, Transfection, Expressing, Western Blot, Control

Journal: bioRxiv

Article Title: The myokine FGF21 associates with enhanced survival in ALS and mitigates stress-induced cytotoxicity

doi: 10.1101/2024.09.11.611693

Figure Lengend Snippet: (A) The viability of NSC-34 cells expressing FLAG-tagged WT-SOD1 or SOD1 G93A was determined using the Vialight assay. Viability for WT SOD1-transfected cells was set at 1. **** P < 0.0001, unpaired two-tailed t-test. (B) Caspase activation was measured in the same cells and values were normalized to activity in WT SOD1-transfected cells which was set at 1. **** P < 0.0001, unpaired two-tailed t-test. (C) FGF21 protein in the conditioned media of NSC-34 cells transfected with a FLAG-tagged FGF21 plasmid was detected by western blot (upper panel) and by ELISA (graph). **** P < 0.0001; unpaired two-tailed t-test. (D) and (E) NSC-34 cells expressing either WT-SOD1 or SOD1 G93A were transfected with FLAG-FGF21 and assessed for viability as in (A) and Caspase-3/7 as in (B). **** P < 0.0001, unpaired two-tailed t-test. (F) Cell viability was assessed in NSC-34 cells exposed to methionine-cystine (MetCys)-deprived media or treated with 100μM H 2 O 2 . **** P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparisons test. (G) NSC-34 cells transfected with FLAG-FGF21 (or empty vector) were subjected to stressors as described in (F) for 24h and then assayed for viability. ** P = 0.007, *** P = 0.0002; unpaired two-tailed t-test. Data points represent biological replicates and bars are the mean ± SD.

Article Snippet: Cells were treated with methionine-cystine (MetCys)-deprived media (Thermo Fisher Scientific) or 100 ng/ml mouse recombinant FGF21 (GenScript) or exposed to 100 μM H 2 0 2 (Sigma Aldrich) for 24 h. Additionally, some cells were transfected with pCDNA3.1 FGF21-Flag (GenScript), Flag-SOD1 G93A and Flag-SOD1WT plasmid constructs using Lipofectamine 2000 (Thermo Fisher Scientific) in Opti-MEM Reduced Serum Media (Thermo Fisher Scientific).

Techniques: Expressing, Transfection, Two Tailed Test, Activation Assay, Activity Assay, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The myokine FGF21 associates with enhanced survival in ALS and mitigates stress-induced cytotoxicity

doi: 10.1101/2024.09.11.611693

Figure Lengend Snippet: (A) C2C12 myoblasts were transfected with FLAG-tagged WT and SOD1 G93A expression plasmids and lysates were assessed by western blot using the antibodies indicated. Bands were quantitated by densitometry and a ratio to the loading control, vinculin, was calculated (shown between the two blots). (B) FGF21 mRNA levels were measured in the same lysates (left panel) and FGF21 protein in the conditioned media was quantified by ELISA (right panel). * P = 0.011, ** P = 0.008; unpaired two-tailed t-test. Secretory FGF21 from the conditioned media was quantified using ELISA (Right panel). ( C) Viability of NSC-34 cells expressing FLAG-tagged WT-SOD1 or SOD1 G93A was determined using the Vialight assay (Left panel). Caspase activation was measured in the same cells and values were normalized to activity in WT SOD1-transfected cells which was set at 1 (right panel). **** P < 0.0001, unpaired two-tailed t-test. (D) FGF21 protein in the conditioned media of C2C12 cells transfected with a FLAG-tagged FGF21 was detected by western blot (upper panel) and by ELISA (graph). Estimated size of the band (kDa) is shown to the right of the blot. **** P < 0.0001; unpaired two-tailed t-test. (E) and (F) C2C12 myoblasts cells expressing either WT-SOD1 or SOD1 G93A were transfected with FGF21-FLAG and assessed for viability and Caspase-3/7 activity as in (C). ** P = 0.003, *** P = 0.0003; unpaired two-tailed t-test. (G) FGF21 mRNA levels were quantified in C2C12 cells exposed to MetCys-deprived media or treated with 100μM H 2 O 2 for 24 h. **** P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparisons test. (H) Cell viability was assessed in C2C12 myoblasts exposed to stressors as described in (G). **** P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparisons test. (I) C2C12 cells transfected with FGF21-FLAG (or empty vector) were subjected to stressors as described in (G) for 24h and then assayed for viability. **** P < 0.0001; unpaired two-tailed t-test. Data points represent biological replicates and bars are the mean ± SD.

Article Snippet: Cells were treated with methionine-cystine (MetCys)-deprived media (Thermo Fisher Scientific) or 100 ng/ml mouse recombinant FGF21 (GenScript) or exposed to 100 μM H 2 0 2 (Sigma Aldrich) for 24 h. Additionally, some cells were transfected with pCDNA3.1 FGF21-Flag (GenScript), Flag-SOD1 G93A and Flag-SOD1WT plasmid constructs using Lipofectamine 2000 (Thermo Fisher Scientific) in Opti-MEM Reduced Serum Media (Thermo Fisher Scientific).

Techniques: Transfection, Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Activation Assay, Activity Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: The myokine FGF21 associates with enhanced survival in ALS and mitigates stress-induced cytotoxicity

doi: 10.1101/2024.09.11.611693

Figure Lengend Snippet: (A) C2C12 myoblasts were treated with DM for various time intervals and immunostained with an anti-MHC antibody followed by DAPI counterstaining. Myotube formation was detected by MHC-positive staining. Scale bars, 100 µM. The fusion index (%) was quantified as described in the methods (right panel). (B) C2C12 myoblasts treated with DM were lysed at specific time intervals and immunoblotted with antibodies against MHC and vinculin. Densitometry values (24 h time interval was set at 1) are shown. (C) FGF21 mRNA levels were quantified from the lysates and FGF21 protein from conditioned media. *** P = 0.0005 comparing to the 24 h time interval, #### P < 0.0001 comparing to the 24 and 48 h time intervals; one-way ANOVA followed by Tukey’s multiple comparisons test. (D) C2C12 myoblasts were transfected with an FGF21-FLAG plasmid and cultured in DM for 96 h. Myotube formation was assessed by MHC-positive staining as in (A). Scale bar, 100 µM. (E) Fusion index for transfected C2C12 cells. ** P = 0.008; unpaired two-tailed t-test. (F) Immunoblot analysis for MHC and vinculin was performed as described in (B). (G) FGF21 levels in the conditioned media from C2C12 myoblasts transfected with FGF21-FLAG or empty vector control were quantified by ELISA (upper graph). **** P < 0.0001; unpaired two-tailed t test. Cells were reseeded and cultured in growth medium (GM) for 72 h. Cell proliferation was assessed at indicated time intervals using MTS (lower graph). ** P = 0.007, **** P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparisons test. Data points represent biological replicates and bars are the mean ± SD.

Article Snippet: Cells were treated with methionine-cystine (MetCys)-deprived media (Thermo Fisher Scientific) or 100 ng/ml mouse recombinant FGF21 (GenScript) or exposed to 100 μM H 2 0 2 (Sigma Aldrich) for 24 h. Additionally, some cells were transfected with pCDNA3.1 FGF21-Flag (GenScript), Flag-SOD1 G93A and Flag-SOD1WT plasmid constructs using Lipofectamine 2000 (Thermo Fisher Scientific) in Opti-MEM Reduced Serum Media (Thermo Fisher Scientific).

Techniques: Staining, Transfection, Plasmid Preparation, Cell Culture, Two Tailed Test, Western Blot, Control, Enzyme-linked Immunosorbent Assay