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: Biochemical Journal

Article Title: Circulating FGF21 proteolytic processing mediated by fibroblast activation protein

doi: 10.1042/bj20151085

Figure Lengend Snippet: Figure 2 The levels of FGF21 processed products in healthy volunteer donors

Article Snippet: FGF21 in vitro digestion assays For FAP in vitro digestion of FGF21, human or mouse FGF21 was incubated at 37 ◦C with recombinant FAP (R&D Systems) at a ratio of 10:1 (FGF21:FAP) in digestion buffer (25 mM Tris, 0.25 M NaCl, pH 8.0).

Techniques:

Journal: Biochemical Journal

Article Title: Circulating FGF21 proteolytic processing mediated by fibroblast activation protein

doi: 10.1042/bj20151085

Figure Lengend Snippet: Figure 5 Human FGF21 cleavage in mouse plasma from wild-type and FAP knockout mice

Article Snippet: FGF21 in vitro digestion assays For FAP in vitro digestion of FGF21, human or mouse FGF21 was incubated at 37 ◦C with recombinant FAP (R&D Systems) at a ratio of 10:1 (FGF21:FAP) in digestion buffer (25 mM Tris, 0.25 M NaCl, pH 8.0).

Techniques: Clinical Proteomics, Knock-Out

Journal: Biochemical Journal

Article Title: Circulating FGF21 proteolytic processing mediated by fibroblast activation protein

doi: 10.1042/bj20151085

Figure Lengend Snippet: Figure 4 FGF21 processing in plasma with FAP inhibitor or FAP immunodepleted

Article Snippet: FGF21 in vitro digestion assays For FAP in vitro digestion of FGF21, human or mouse FGF21 was incubated at 37 ◦C with recombinant FAP (R&D Systems) at a ratio of 10:1 (FGF21:FAP) in digestion buffer (25 mM Tris, 0.25 M NaCl, pH 8.0).

Techniques: Clinical Proteomics

Journal: Molecular metabolism

Article Title: Fibroblast growth factor 21 has no direct role in regulating fertility in female mice.

doi: 10.1016/j.molmet.2016.05.010

Figure Lengend Snippet: Figure 3: High fat diet reverses the infertility in FGF21-Tg mice. A) Body weight of WT and FGF21-Tg mice fed either with chow or HFD (n ¼ 4e6). B) Body composition as represented as absolute fat and lean mass (n ¼ 6e7). C) Assessment of estrus cycle in FGF21-Tg mice fed either chow or HFD. (1: leukocytes [diestrus/metestrus], 2: cornified cells [estrus], 3: nucleated cells [proestrus]) D) Representative vaginal cytology from FGF21 Tg mice fed with HFD as (i) diestrus, (ii) proestrus, (iii) estrus, (iv) metestrus. Scale: 200 mm (ieiv). (n ¼ 8). E) Ovarian histology from FGF21-Tg mice; (i) chow, (ii) HFD, (iiieiv) HFD fed pregnant females. Corpus lutea (CL) are encircled with dotted lines. Scale: 1000 mm. Statistical significance was evaluated by Student’s t test; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Mouse FGF21 and human FGF21 proteins were measured by ELISA (R & D Systems, MN).

Techniques:

Journal: Molecular metabolism

Article Title: Fibroblast growth factor 21 has no direct role in regulating fertility in female mice.

doi: 10.1016/j.molmet.2016.05.010

Figure Lengend Snippet: Figure 5: Infertility of FGF21-Tg mice is independent of FGF21. Serum levels of total FGF21 (A) and active FGF21 (B) in FGF21-Tg mice fed with chow or HFD (n ¼ 9e10). AVP and Kiss-1 gene expression in SCN (C) and AVPV (D), respectively of WT and FGF21-Tg mice fed chow or HFD (n ¼ 8e10). All mice were in the diestrus phase. Statistical significance was evaluated by Student’s t test; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Mouse FGF21 and human FGF21 proteins were measured by ELISA (R & D Systems, MN).

Techniques: Gene Expression

Journal: Endocrine Connections

Article Title: Exercise ameliorates the FGF21–adiponectin axis impairment in diet-induced obese mice

doi: 10.1530/EC-19-0034

Figure Lengend Snippet: The effects of exercise on FGF21 and adiponectin levels was determined. (A) Serum levels of FGF21. (B) FGF21 mRNA expression in the liver. (C) mRNA expression of FGF21 in epididymal WAT. (D) Serum levels of total adiponectin. (E) HMW adiponectin concentrations in circulation. (F) Adiponectin mRNA levels in epididymal WAT. Data are presented as the mean ± s.e.m. ; n = 9–10 per group. a P < 0.05 for difference from LFD; b P < 0.05 for difference from HFD.

Article Snippet: To analyze the effects of acute FGF21 intervention on adiponectin secretion, the mice were intraperitoneally administered saline or 2 μg/g ( ) of recombinant human FGF21 (PeproTech Inc., Rocky Hill, NJ, USA).

Techniques: Expressing

Journal: Endocrine Connections

Article Title: Exercise ameliorates the FGF21–adiponectin axis impairment in diet-induced obese mice

doi: 10.1530/EC-19-0034

Figure Lengend Snippet: Exercise improves the ability of FGF21 to induce adiponectin secretion in HFD-induced obese mice. (A) FGF21 stimulates total adiponectin secretion. (B) Fold increase of FGF21-induced total adiponectin secretion. (C) FGF21 increases HMW adiponectin secretion. (D) Fold increase of FGF21-induced HMW adiponectin secretion. Data are presented as the mean ± s.e.m. ; n = 7 per group. For A and C, a P < 0.05, saline administration vs FGF21 administration. For B and D, a P < 0.05 for difference from LFD; b P < 0.05 for difference from HFD.

Article Snippet: To analyze the effects of acute FGF21 intervention on adiponectin secretion, the mice were intraperitoneally administered saline or 2 μg/g ( ) of recombinant human FGF21 (PeproTech Inc., Rocky Hill, NJ, USA).

Techniques: Saline