mouse recombinant fgf21  (PeproTech)

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

Journal: bioRxiv

Article Title: Sulfur Amino Acid Restriction Enhances Exercise Capacity in Mice by Boosting Fat Oxidation in Muscle

doi: 10.1101/2024.06.27.601041

Figure Lengend Snippet: A. Experimental design and color scheme used in and figure S4A-C. B. Percent change in body weight (n = 11 - 24) of male WT or FGF21KO mice given ad libitum access sulfur amino acid restricted (SAAR) versus control (Con) diet for seven days. C. Distance ran during a one-time maximal endurance test (n = 11 - 24) of male WT or FGF21KO mice given ad libitum access to SAAR versus Con diet on day seven. D. Experimental set up and color scheme used throughout E-F and figure S4D-E. E. Percent change in body weight (n = 8) of NaCl or recombinant FGF21 treated male mice for seven days. F. Distance ran during a one-time maximal endurance test (n = 8) of NaCl or recombinant FGF21 treated male mice on day seven. All data is shown as mean and error bars indicate SD unless otherwise noted; p values indicate the significance of the difference by Student’s t test between treatments, or two-way ANOVA with Sidak’s multiple comparisons test between diets and genotype; significance is determined by a p value of p < 0.05. See also Figure S4.

Article Snippet: Mouse recombinant FGF21 (Cat# 450-56, Peprotech) was dissolved and diluted in sterile distilled water to a final dosage of 1 mg/kg/day.

Techniques: Control, Recombinant

Journal: Nature Communications

Article Title: Short-term hypercaloric carbohydrate loading increases surgical stress resilience by inducing FGF21

doi: 10.1038/s41467-024-44866-3

Figure Lengend Snippet: a Serum FGF21 levels after one week of preconditioning with control or low protein diet (LP) with control (Ctrl) or 50% sucrose-water (HC). b Relative liver Fgf21 mRNA levels after one week of preconditioning with the indicated diet. c Relative kidney Fgf21 mRNA levels after one week of preconditioning with the indicated diet. d Linear regression showing the relationship between serum FGF21 levels versus protein intake and (e) serum urea (AUC). R 2 coefficient was calculated using Pearson’s method. f Serum FGF21 levels after one week of preconditioning with Ctrl, HC or HC with cysteine oral gavage (HC + Cysteine). g Volcano plot of liver differential expressed genes (DEG) with FDR < 0.05 and log2FC > 2 from FGF21 overexpressing male mice from GSE39313. h Fold change of top DEGs from GSE39313 in liver after one week of preconditioning with HC compared to Ctrl. * p values of a,b were calculated with two-way ANOVA followed by a Tukey’s post hoc analysis, d,e are linear regressions and r squared Pearson’s coefficient, f with one-way ANOVA and h with unpaired two-tailed T-test with Bonferroni correction, * p < 0.05 * *p < 0.01 *** p < 0.001 **** p < 0.0001. p- values for a = 0.0001, for b = 0.0001, for d < 0.0001, for f < 0.0001, for h = 0.0003. In all panels, experiments were carried out in 10-weeks old male mice. Sample sizes: ( a–c ), n = 8; ( d-e) , n = 32; (f ), n = 5 for all conditions; ( h ), n = 8 for all conditions. Data in all panels are shown as mean ± SD. See also Supplementary Data Figs. and .

Article Snippet: Mouse recombinant FGF21 (Cat# 450-56, Peprotech) was dissolved and diluted in sterile distilled water to a final dosage of 1 mg/kg/day.

Techniques: Control, Two Tailed Test

Journal: Nature Communications

Article Title: Short-term hypercaloric carbohydrate loading increases surgical stress resilience by inducing FGF21

doi: 10.1038/s41467-024-44866-3

Figure Lengend Snippet: a Serum FGF21 levels after one week of preconditioning with standard (Ctrl) or 50% sucrose-water (HC). b Food (left) and water (right) intake (normalized by body weight) after one week of preconditioning with the indicated diet. c Serum urea (AUC) and ( d ) serum creatinine levels at day 2 post-renal IRI after one week of preconditioning with the indicated diet. e Representative cross sections of PAS-stained kidneys (left; x10 magnification; scale bar 100 µm) and histological score (right) at day 2 post-renal IRI after one week of preconditioning with the indicated diet. f Representative cross sections of PAS-stained kidneys with necrotic area digitally highlighted in red (left; x10 magnification; scale bar 100 µm) and quantification of necrotic tissue area (right) at day 2 post-renal IRI after one week of preconditioning with the indicated diet. g Relative Krt20 mRNA levels in the kidney at day 2 post-renal IRI after one week of preconditioning with the indicated diet. h Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzyme levels at the indicated time post-hepatic IRI. i Representative cross sections of H&E-stained livers (x20 magnification; scale bar 30 µm) at day 1 post-hepatic IRI. Ischemic areas appearing in light pink. j Relative Hmgb1 mRNA levels in the liver at day 2 post-renal IRI after one week of preconditioning with the indicated diet. * p values for a,c-g,j were calculated with two-way ANOVA followed by a Tukey’s post hoc analysis, b with two-way RM ANOVA with Geisser-Greenhouse correction and h with two-way ANOVA followed by a Sidak’s post hoc analysis, * p < 0.05 ** p < 0.01 *** p < 0.001 **** p < 0.0001 a p < 0.05 aaaa p < 0.0001 Fgf21 WT HC vs. Fgf21 WT Ctrl, bbbb p < 0.0001 Fgf21 KO HC vs. Fgf21 WT Ctrl. p values for a < 0.0001, <0.0001 and <0.0001, for b < 0.0001 and <0.0001, for c = 0.0269 and 0.0008, for d = 0.0453, for e = 0.0236 and 0.001, for f < 0.0001, <0.0001 and = 0.0074, for g = 0.0002 and 0.0031, for h = 0.0328 and 0.0351, for j = 0.007 and 0.022. In a-j , experiments were carried out in Fgf21 WT and Fgf21 KO 10-weeks old male mice. Sample sizes: ( a–g) , n = 10–15, n = 15 for Fgf21 WT Ctrl, n = 15 for Fgf21 WT HC, n = 15 Fgf21 KO Ctrl, n = 10 for Fgf21 KO HC; ( h–j ), n = 6–13, n = 6 Fgf21 WT Ctrl and HC, n = 10 for Fgf21 KO Ctrl, n = 13 for Fgf21 KO HC. Data in all panels are shown as mean ± SD. See also Supplementary Data Fig. .

Article Snippet: Mouse recombinant FGF21 (Cat# 450-56, Peprotech) was dissolved and diluted in sterile distilled water to a final dosage of 1 mg/kg/day.

Techniques: Staining

Journal: Nature Communications

Article Title: Short-term hypercaloric carbohydrate loading increases surgical stress resilience by inducing FGF21

doi: 10.1038/s41467-024-44866-3

Figure Lengend Snippet: a Experimental setup. Ad-libitum fed 10-weeks-old male mice were given free access to a standard diet (Ctrl) and implanted with osmotic pumps containing either NaCl (Veh) or human recombinant FGF21 (FGF21; 1 mg/kg/day) for 7 days prior to renal IRI surgery. Created with BioRender.com. b Serum FGF21 levels (left) and AUC (right) post-renal IRI after one week of preconditioning with the indicated treatment. c Food (left) and water (right) intake (normalized by body weight) after one week of preconditioning with the indicated treatment. d Serum urea levels (left) and AUC (right) at the indicated time post-renal IRI. e Serum creatinine levels at day 2 post-renal IRI after one week of preconditioning with the indicated treatment. f Relative Krt20 mRNA levels at day 2 post-renal IRI after one week of preconditioning with the indicated treatment. g Representative cross sections of PAS-stained kidneys (left; x10 magnification; scale bar 100 µm) at day 2 post-renal IRI and histological score (right) after one week of preconditioning with the indicated treatment. h Representative cross sections of PAS-stained kidneys with necrotic area digitally highlighted in red and quantification (left; x10 magnification; scale bar 100 µm) and necrotic tissue area (right) at day 2 post-renal IRI after one week of preconditioning with the indicated treatment. * p values for b,c were calculated with two-way RM ANOVA with Geisser-Greenhouse correction, c with two-way ANOVA followed by a Sidak’s post hoc analysis, and b,d,e,f,g,h with unpaired two-tailed T-tests, * p < 0.05 ** p < 0.01 *** p < 0.001 **** p < 0.0001. p values for b < 0.0001 and <0.0001, for c = 0.0120, 0.0002, 0.0028, 0.0121, 0.0129 and 0.0001, for d = 0.0667, for e = 0.0022, for f = 0.0129, for g = 0.0488, for h = 0.0471. Image1A was partially created with BioRender.com. In b-h , experiments were carried out in 10-week-old male mice. Sample sizes: ( a–h ), n = 6-8, n = 8 Ctrl and n = 6 FGF21 treated group. Data in all panels are shown as mean ± SD. See also Supplementary Data Fig. .

Article Snippet: Mouse recombinant FGF21 (Cat# 450-56, Peprotech) was dissolved and diluted in sterile distilled water to a final dosage of 1 mg/kg/day.

Techniques: Recombinant, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Short-term hypercaloric carbohydrate loading increases surgical stress resilience by inducing FGF21

doi: 10.1038/s41467-024-44866-3

Figure Lengend Snippet: a Module-Trait relationships with serum FGF21 levels identified through Weighted Gene Co-expression Network Analysis (WGCNA) from kidneys of male mice given access to ad libitum Ctrl, HC, LP and LPHC diet for one week. Top line corresponds to Pearson R 2 and bottom line to adjusted p value. Color scales represent positive correlation (red) and negative correlation (blue). b Module-Trait relationships with serum FGF21 levels identified through WGCNA from livers of mice after one week on diets with 18%, 14%, 10%, 6%, 2% and 0% protein content. Top line corresponds to Pearson R 2 and bottom line to adjusted p value. c Common biological processes (GO terms) enriched in kidney and liver modules with significant positive and ( d ) negative Module-Trait relationships with serum FGF21 identified through WGCNA. The dot size corresponds to the number of genes in the pathway. The color of the dots indicates the degree of significance of the pathway enrichment, with darker shades indicating higher significance. e Heatmap of percentile-transformed expression levels of the top 15 genes from kidney and liver ( f ) modules with significant Module-Trait relationships with serum FGF21. Yellow indicating high expression and blue indicating low expression. The genes from each module with positive Module-Trait relationships with serum FGF21 were used in STRING and non-singleton were used for transcription factor analysis (TFA). The motifs picture shows the top NES motifs and their associated direct transcription factors. g Average mRNA expression levels of genes from kidney with slope > 10 and R 2 > 0.70 for each diet group (Ctrl, HC, LP, and LP+HC). Top panel shows genes with positive slopes, while bottom panel shows negative slopes, reflecting protein dilution intake. h Heatmap of percentile-transformed expression levels of the top 20 genes from kidney with slope > 10 and R squared > 0.70 for each diet group (Ctrl, HC, LP, and LP+HC). Yellow indicates high expression and blue indicates low expression. Upregulated genes were used in STRING and nonsingleton were used for transcription factor analysis (TFA). The motifs picture shows the top NES motifs and their associated direct transcription factors. p values for a and b were calculated with Pearson correlation, and c and d with Benjamin-Hochberg (BH) test with FDR < 0.05. Sample sizes: ( g ), n = 3–6, n = 3 Ctrl, n = 3 HC, n = 6 LP, n = 5 LP + HC. See also Supplementary Data Fig. and .

Article Snippet: Mouse recombinant FGF21 (Cat# 450-56, Peprotech) was dissolved and diluted in sterile distilled water to a final dosage of 1 mg/kg/day.

Techniques: Expressing, Transformation Assay