Journal: Scientific Reports

Article Title: Fgf21 regulates T-cell development in the neonatal and juvenile thymus

doi: 10.1038/s41598-017-00349-8

Figure Lengend Snippet: Fgf21 mRNA is enriched in mature mTECs. ( A and B ) Expression levels of Fgf21 mRNA in the various tissues from 4-week-old C57BL/6 mice ( A ) and in the thymus of E15.5 and 0-, 4-, 8- and 12-week-old ( B ). Relative Fgf21 mRNA expression was normalised against 18S rRNA expression. ( C and D ) Thymic cell suspensions from 4-week-old C57BL/6 mice were assessed by flow cytometric analysis using anti-CD45 mAb, anti-EpCAM mAb, anti-MHC II (I-A/I-E) mAb, and UEA-1 lectin, and expression levels of Fgf21 mRNA were determined by RT-realtime PCR in the subpopulations of thymic cells from 4-week-old C57BL/6 mice. ( C ) Anti-CD45 and anti-EpCAM staining discriminated CD45 + , TEC (CD45 − EpCAM + ), and non-TEC stromal cell (CD45 − EpCAM − ) subpopulations. ( D ) Gated TECs were subdivided into cTEC hi (UEA-1 − MHC high ), cTEC lo (UEA-1 − MHC low ), mTEC hi (UEA-1 + MHC high ), and mTEC lo (UEA-1 + MHC low ) subpopulations. ( E ) Thymocytes were removed from embryonic thymi by 1.35 mM dGuo treatment for 6 days, and remaining stromal cells were stimulated with 1 μg/ml RANKL to induce the maturation of TECs. Fgf21 and Aire mRNA were increased along with TEC maturation. Graphs represent the mean ± SD; n ≧5 replicates per group from at least 2 independent experiments.

Article Snippet: Medium containing 500 ng/ml of recombinant human FGF21 (R&D Systems) was replaced at 3-day intervals.

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: Fgf21 regulates T-cell development in the neonatal and juvenile thymus

doi: 10.1038/s41598-017-00349-8

Figure Lengend Snippet: CD4SP and CD8SP populations are decreased in Fgf21 KO mice. ( A and B ) Thymi and spleens were isolated from 4-week-old WT and Fgf21 KO mice, and their weights were measured. ( C ) Cell numbers of enzymatically digested suspensions from thymi and spleens were counted. ( D ) Thymocytes and splenocytes from WT and Fgf21 KO mice were stained with anti-CD4 and anti-CD8 mAb. Representative flow cytometry plots show CD4/CD8 analysis of thymocytes and splenocytes from 1-week-old WT and Fgf21 KO mice. ( E ) Charts show the percentage of DN, DP, CD8SP, and CD4SP cells from 1- and 4-week-old WT and Fgf21 KO mice. ( F ) Charts show the percent of CD4SP and CD8SP cells from 1- and 4-week-old WT and Fgf21 KO mice. ( G ) Thymocytes from 1-week-old WT and Fgf21 KO mice were defined by TCRβ and CD69 levels and subdivided into 5 subsets (T1; TCRβ − CD69 − , T2; TCRβ int CD69 − , T3; TCRβ int CD69 + , T4; TCRβ hi CD69 + , and T5; TCRβ hi CD69 − ). ( H ) Charts show the percentage of the 5 subsets from 1-week-old WT and Fgf21 KO mice. ( I ) Gated CD4SP and CD8SP cells from 1-week-old WT and Fgf21 KO mice were defined by CD62L and CD24 levels and subdivided into CD24 + CD62L − immature and CD24 − CD62L + mature SP cells. ( J ) Charts show the percentage of CD24 + CD62L − immature and CD24 − CD62L + mature SP subsets from 1-week-old WT and Fgf21 KO mice. All data shown are the mean ± SD from ≧6 mice per genotype from 2 independent experiments. *P < 0.05, *P < 0.01 versus WT mice.

Article Snippet: Medium containing 500 ng/ml of recombinant human FGF21 (R&D Systems) was replaced at 3-day intervals.

Techniques: Isolation, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: Fgf21 regulates T-cell development in the neonatal and juvenile thymus

doi: 10.1038/s41598-017-00349-8

Figure Lengend Snippet: Thymic stromal cells are not obviously altered in Fgf21 KO mice. ( A ) Enzymatically digested thymic cell suspensions were stained with anti-CD45 mAb and anti-EpCAM mAb, and the TEC (CD45 − EpCAM + ) number was counted from the thymi of E14.5, E15.5, E18.5, and 4-week-old WT and Fgf21 KO mice. ( B ) Immunohistochemistry of WT and Fgf21 KO thymi with anti-Keratin-5 antibody for a medullary TEC marker (left panels) and anti-Ly-51 antibody for a cortical TEC marker (right panels). ( C ) The ratio of Keratin-5-positive thymic medullary regions was measured from immunostained sections. ( D ) Gated TECs of 1-weeks old were subdivided into mTEC hi (Ly51 − MHC high ), mTEC lo (Ly51 − MHC low ), cTEC hi (Ly51 + MHC high ), and cTEC lo (Ly + MHC low ) subpopulations. Graphs represent the percentage of subpopulations. ( E ) The mean fluorescence intensity (MFI) of surface MHCII on the TEC was measured by flow cytometry with anti-MHCII antibody. ( F ) Gated CD45 − thymic stromal cells of 1-weeks old were stained with anti-CD140a and anti-CD31 antibodies. Graphs represent the cellularity of CD45 − CD140a + fibroblasts and CD45 − CD31a + endothelial cells in the thymus of WT and Fgf21 KO mice. ( G ) The expression levels of thymic factors were measured in the thymus of 1-week-old WT and Fgf21 KO mice. Graphs represent the mean ± SD; n ≧ 5 replicates per group from at least 3 independent experiments.

Article Snippet: Medium containing 500 ng/ml of recombinant human FGF21 (R&D Systems) was replaced at 3-day intervals.

Techniques: Staining, Immunohistochemistry, Marker, Fluorescence, Flow Cytometry, Expressing

Journal: Scientific Reports

Article Title: Fgf21 regulates T-cell development in the neonatal and juvenile thymus

doi: 10.1038/s41598-017-00349-8

Figure Lengend Snippet: FGF21 treatment alters T-cell development in FTOC. WT and Fgf21 KO foetal thymi were cultured with or without recombinant human FGF21 protein (500 ng/ml) for 14 days. ( A ) Total cell numbers of thymocytes in FTOCs are presented. Flow cytometry analysis of CD4/8 ( B ) and TCRβ/CD69 ( C ) distribution and thymocyte subset number ( D and E ). ( F ) Representative flow cytometry data for each FTOC. ( G ) Numbers of mTEC (CD45 − EpCAM + UEA-1 + ), cTEC (CD45 − EpCAM + UEA-1 − ), and non-TEC stromal cells (CD45 − EpCAM − ) are shown. Data represents the mean ± SD of two separate experiments. *,# P < 0.05, **,## P < 0.01, and *** P < 0.001 versus WT without rhFGF21, $ P < 0.05, and $$ P < 0.01 versus Fgf21 KO without rhFGF21.

Article Snippet: Medium containing 500 ng/ml of recombinant human FGF21 (R&D Systems) was replaced at 3-day intervals.

Techniques: Cell Culture, Recombinant, Flow Cytometry

Journal: Scientific Reports

Article Title: Fgf21 regulates T-cell development in the neonatal and juvenile thymus

doi: 10.1038/s41598-017-00349-8

Figure Lengend Snippet: FGF21 treatment results in increased apoptosis of immature thymocytes. FTOC was performed in the presence or absence of recombinant human FGF21 (500 ng/ml) for 14 days. Annexin V staining was performed to detect apoptotic T cells by flow cytometry. ( A ) Representative histograms show the percentage of Annexin V + cells in gated thymocyte subsets. ( B ) Graph showing the analysis of the percentage of Annexin V + cells in thymocytes from FTOCs. All data shown are the mean ± SD. **P < 0.01 versus control.

Article Snippet: Medium containing 500 ng/ml of recombinant human FGF21 (R&D Systems) was replaced at 3-day intervals.

Techniques: Recombinant, Staining, Flow Cytometry, Control

Journal: Scientific Reports

Article Title: Fgf21 regulates T-cell development in the neonatal and juvenile thymus

doi: 10.1038/s41598-017-00349-8

Figure Lengend Snippet: Fgf21 induced by protein-free diet does not affect thymic change by protein malnutrition. ( A ) Serum Fgf21 levels of 4-week-old C57BL/6 mice fed for 1 week with normal chow diet (NC) or protein-free diet (PF) were determined with ELISA assays. Data shown are the mean ± SD from ≧6 mice. ** P < 0.001 versus NC. ( B ) Relative expression levels of Fgf21 mRNA in the liver, thymus, subcutaneous white adipose tissue (sWAT), and skeletal muscle from mice fed with NC or PF. Data shown are the mean ± SD from ≧6 mice. ** P < 0.01, ** P < 0.001 versus NC. ( C–F ) Body weight ( C ), blood glucose ( D ), thymic weight ( E ), and thymic cell number ( F ) of WT and Fgf21 KO mice fed with NC or PF. Data shown are the mean ± SD from ≧5 mice. * P < 0.05, # P < 0.05 versus WT and Fgf21 KO mice fed with NC, respectively. $ P < 0.05 versus WT mice fed with PF. ( G and H ) Thymocytes from WT and Fgf21 KO mice fed with NC or PF were stained with anti-CD4 and anti-CD8 mAb ( G ), or anti-TCRβ and anti-CD69 mAb ( H ). All data shown are the mean ± SD from ≧5 mice. * P < 0.05, # P < 0.05 versus WT and Fgf21 KO mice fed with NC, respectively. $ P < 0.05 versus WT mice fed with PF.

Article Snippet: Medium containing 500 ng/ml of recombinant human FGF21 (R&D Systems) was replaced at 3-day intervals.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Staining

Journal: Cell Metabolism

Article Title: GDF15 Provides an Endocrine Signal of Nutritional Stress in Mice and Humans

doi: 10.1016/j.cmet.2018.12.016

Figure Lengend Snippet: GDF15 Expression Is Regulated by the Cellular ISR Pathway (A and C) GDF15 mRNA expression (A) and immunoblot analysis (C) of ISR components in wild-type (WT) mouse embryonic fibroblasts (MEFs) treated with vehicle control (Con), cobalt chloride (CoCl2, 625 μM), thapsigargin (Tg, 1 μM), tunicamycin (Tn 5 μg/mL), or L-Histidinol (His, 1 mM) for 6 h. (B) GDF15 mRNA expression in human cell lines (HeLA, HuH7, and A549) treated with Tn (5 μg/mL) for 6 h. (D) GDF15 mRNA expression in WT MEFs pre-treated for 1 h either with the PERK inhibitor GSK2606414 (GSK, 200 nM) or eIF2α inhibitor ISRIB (ISR, 100 nM), then co-treated with Tn (5 μg/mL) for a further 6 h. (E–G) GDF15 mRNA expression (E) in EIF2α Ser51 (SS) or phospho mutant (AA) MEFs or (F) in ATF4 wild-type (WT) or ATF4 knockout (KO) MEFs and (G) in control siRNA and CHOP siRNA transfected WT MEFs treated with Tn (5 μg/mL) for 6 h. (H) Diagram outlining pathway by which GDF15 and FGF21 expression is regulated by TN. mRNA expression is presented as fold expression relative to its respective control treatment for each cell type (set at 1) or TN-treated samples (set as 100) with normalization to HPRT gene expression in MEFs and GAPDH in human cells. Data are expressed as mean ± SD from at least three independent experiments. ∗∗∗ p < 0.001 versus control (con) for (A) and (B), and versus TN stimulated for (D)–(G) by two-tailed Student’s t test. Blots shown are representative of three independent experiments with Calnexin used as a loading control. See also Figures S3 and .

Article Snippet: Mouse FGF21 was analyzed by FGF21 Quantakine ELISA (R&D Systems) following the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Control, Mutagenesis, Knock-Out, Transfection, Gene Expression, Two Tailed Test

Journal: Cell Metabolism

Article Title: GDF15 Provides an Endocrine Signal of Nutritional Stress in Mice and Humans

doi: 10.1016/j.cmet.2018.12.016

Figure Lengend Snippet:

Article Snippet: Mouse FGF21 was analyzed by FGF21 Quantakine ELISA (R&D Systems) following the manufacturer’s instructions.

Techniques: Recombinant, SYBR Green Assay, Protease Inhibitor, Reverse Transcription, Western Blot, Injection, Enzyme-linked Immunosorbent Assay, Control, TaqMan Assay, Software, Sterility, Electrophoresis, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: FoxO1 suppresses Fgf21 during hepatic insulin resistance to impair peripheral glucose utilization and acute cold tolerance

doi: 10.1016/j.celrep.2021.108893

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Serum concentrations FGF21 and insulin were determined by ELISA assay using commercial kits (FGF21: R&D Systems, cat#MF2100; insulin: Chrystal Chem, cat#90080) according to the manufacturers’ instructions.

Techniques: Recombinant, Injection, Modification, SYBR Green Assay, Electron Microscopy, Enzyme-linked Immunosorbent Assay, Double Knockout, Triple Knockout, Isolation, Real-time Polymerase Chain Reaction, Plasmid Preparation, Blocking Assay, Software, Imaging, Microscopy