Journal: Nature

Article Title: PTER is a N -acetyltaurine hydrolase that regulates feeding and obesity

doi: 10.1038/s41586-024-07801-6

Figure Lengend Snippet: a , b , Change in body weight ( a ) and food intake ( b ) of 26–28-week-old male DIO C57BL/6J mice following 7 days of treatment with the indicated dose of N -acetyltaurine (NAT; i.p.). For saline versus N -acetyltaurine (15 mg per kg per day), P = 5.95 × 10 –4 ; for saline versus N -acetyltaurine (50 mg per kg per day), P = 6.3 × 10 –4 . N = 5 per group for vehicle, 1 and 5 mg per kg per day; N = 6 per group for 15 and 50 mg per kg per day. c , d , Change in body weight ( c ) and food intake ( d ) of 19–21-week-old male DIO C57BL/6J mice following treatment with the indicated metabolite at a dose of 15 mg per kg per day (i.p.). N = 5 per group. e – g , Western blots with anti-PTER (top) and anti-tubulin (bottom) antibodies ( e ), N -acetyltaurine hydrolysis activity ( f ) and tissue N -acetyltaurine levels ( g ) from cortex (Cort.), hypothalamus (Hyp.) and brainstem (BS) of WT mice and Pter KO mice. For WT versus Pter KO brainstem, P = 6.65 × 10 –4 . N = 6 per group for f and g . h , Change in 24-h food intake of 6-month-old male DIO mice treated with a single dose of GDF15 (0.1 mg kg –1 , i.p.) in the presence of anti-GFRAL antibody (10 mg kg –1 , i.p.) or IgG control antibody (10 mg kg –1 , i.p.). N = 5 per group. i , j , Change in body weight ( i ) and cumulative food intake ( j ) of 16-week-old male DIO mice following saline or N -acetyltaurine (15 mg per kg per day, i.p.) treatment and with IgG or anti-GFRAL antibody co-treatment (10 mg kg –1 , i.p., once every 3 days). N = 10 per group. Data are shown as the mean ± s.e.m. For e , the loading control was performed on the same blot. In a – d and f – h , P values were calculated from two-tailed unpaired t -tests and were not corrected for multiple comparisons. In i and j , P values were calculated from two-way ANOVA with post hoc Sidak’s multiple comparisons test. All experiments were performed once.

Article Snippet: Recombinant GDF15 (957-GD) was purchased from R&D Systems.

Techniques: Saline, Western Blot, Activity Assay, Control, Two Tailed Test

Journal: Nature

Article Title: PTER is a N -acetyltaurine hydrolase that regulates feeding and obesity

doi: 10.1038/s41586-024-07801-6

Figure Lengend Snippet: Related to Fig. . a - d , Body weight ( a , b ) and food intake ( c , d ) of 6 to 7-month-old DIO male C57BL/6J mice after a 7-day treatment of saline or N-acetyltaurine (15 mg/kg/day, IP) or GLP-1 (2 mg/kg/day, IP) with or without Exendin-3 (0.1 mg/kg/day, IP). N = 7/group. NAT, N-acetyltaurine. e , f , Body weight ( e ) and food intake ( f ) of 5-month-old DIO male C57BL/6J mice or 3 to 4-month-old MC4R-KO mice on high fat diet after a 7-day treatment of saline or N-acetyltaurine (15 mg/kg/day, IP). N = 6/group. NAT, N-acetyltaurine. g , Plasma GDF15 (left), GLP-1 (middle) and leptin (right) levels of 19 to 21-week-old male DIO C57BL/6J mice following treatment with N-acetyltaurine 15 mg/kg/day (IP) or saline. N = 5 per group. NAT, N-acetyltaurine. Data are shown as mean ± SEM. P-values were calculated from two-tailed unpaired t-tests.

Article Snippet: Recombinant GDF15 (957-GD) was purchased from R&D Systems.

Techniques: Saline, Clinical Proteomics, Two Tailed Test

Journal: Clinical and Experimental Medicine

Article Title: GDF15 activates human fibroblast MRC5 cells via miR-338/STAT1 in silicosis

doi: 10.1007/s10238-025-01627-w

Figure Lengend Snippet: Increased blood GDF15 expression in patients with silicosis. A , Serum GDF15 expression in patients with silicosis was higher than that in controls ( P = 0.0003, compared to control). B , Increased serum GDF15 expression did not relate to the FEV1% of patients with silicosis; P = 0.2002. C , Increased serum GDF15 expression did not relate to the clinical stages of silicosis, P = 0.5979

Article Snippet: Recombinant human GDF15 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Control

Journal: Clinical and Experimental Medicine

Article Title: GDF15 activates human fibroblast MRC5 cells via miR-338/STAT1 in silicosis

doi: 10.1007/s10238-025-01627-w

Figure Lengend Snippet: GDF15 activates human embryonic lung fibroblast MRC5 cells. The effect of GDF15 on col1a and α-SMA protein expression in MRC5 cells was detected using western blotting. A , dosage course. B , time course. C , GDF15 increased the proliferation of MRC cells, as detected in the CCK-8 assay. D, GDF15 increased the migration of MRC cells, which was detected in the wound healing assay. Data represent the mean ± SD from three independent experiments.*,**, ♯ , P < 0.05

Article Snippet: Recombinant human GDF15 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, CCK-8 Assay, Migration, Wound Healing Assay

Journal: Clinical and Experimental Medicine

Article Title: GDF15 activates human fibroblast MRC5 cells via miR-338/STAT1 in silicosis

doi: 10.1007/s10238-025-01627-w

Figure Lengend Snippet: Effects of GDF15 on the miRNA and mRNA expression in MRC5 cells. Effect of GDF15 on the miRNA profile of MRC5 cells. A , heatmap; B , Volcano plots; C , KEGG analysis. Effect of GDF15 on the mRNA profile of MRC5 cells. D , heatmap; E, Volcano plots; F, KEGG analysis

Article Snippet: Recombinant human GDF15 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing

Journal: Clinical and Experimental Medicine

Article Title: GDF15 activates human fibroblast MRC5 cells via miR-338/STAT1 in silicosis

doi: 10.1007/s10238-025-01627-w

Figure Lengend Snippet: GDF15 reduced the expression of miR-338 in MRC5 cells. A , qRT-PCR results showed that the expression of miR-338 was decreased in response to GDF15 expression. B , Western blotting results showed that miR-338 mimics attenuated the expression of col1a and α-SMA regardless of treatment with GDF15, whereas miR-338 inhibitors promoted the expression of col1 and α-SMA regardless of treatment with GDF15. C , CCK-8 assay results showed that miR-338 mimics reduced the growth rate and the miR-338 inhibitor increased the growth rate of MRC5 cells. Data represent the mean ± SD from three independent experiments.*,**, ♯ , P < 0.05

Article Snippet: Recombinant human GDF15 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay

Journal: Clinical and Experimental Medicine

Article Title: GDF15 activates human fibroblast MRC5 cells via miR-338/STAT1 in silicosis

doi: 10.1007/s10238-025-01627-w

Figure Lengend Snippet: GDF15 increased STAT1 expression in MRC5 cells. A , Western blotting results showed that GDF15 induced STAT1 expression. B , Western blotting results showed that STAT1 siRNA attenuated the expression of col1a and α-SMA, whereas STAT1 cDNA promoted their expression in the presence or absence of GDF15 treatment. C , CCK-8 assay results showed that STAT1 siRNA reduced the growth rate, whereas STAT1 cDNA increased the growth rate of MRC5 cells. Data represent the mean ± SEM from three independent experiments.*,**, ♯ , P < 0.05

Article Snippet: Recombinant human GDF15 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, CCK-8 Assay

Journal: Clinical and Experimental Medicine

Article Title: GDF15 activates human fibroblast MRC5 cells via miR-338/STAT1 in silicosis

doi: 10.1007/s10238-025-01627-w

Figure Lengend Snippet: GDF15 activated MRC5 cells through the miR-338/STAT1 pathway. A , Using TargetScan ( http://www.targetscan.org/ ), the conserved miR-338 binding site in the 3’-UTR of STAT1 mRNA was constructed in the pmirGLO dual-luciferase miRNA target expression vector. Luciferase activity was analyzed in the MRC5 cells. MRC5 cells were co-transfected with miR-30 mimics and a luciferase reporter. B , miR-338 regulated STAT1 protein expression. C , miR-338 mimics attenuated the expression of col1 and α-SMA protein induced by STAT1 overexpression upon co-treatment with GDF15. D, STAT1 knockdown attenuated col1 and α-SMA protein expression induced by the miR-338 inhibitor upon co-treatment with GDF15. Data represent the means ± SD from three independent experiments. *,**, ♯ , ♯♯ , P < 0.05

Article Snippet: Recombinant human GDF15 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay, Construct, Luciferase, Expressing, Plasmid Preparation, Activity Assay, Transfection, Over Expression, Knockdown

Journal: Oncotarget

Article Title: GDF15 contributes to radioresistance and cancer stemness of head and neck cancer by regulating cellular reactive oxygen species via a SMAD-associated signaling pathway

doi: 10.18632/oncotarget.13649

Figure Lengend Snippet: A. Administration of rhGDF15 led to radioresistance in two HNC cell lines. Detroit and KB cells were treated with 20 ng/ml rhGDF15 for 5 days and then subjected to various doses of irradiation (0, 2, and 4 Gy). Fourteen days later, the cell colonies were determined by staining with 0.05% crystal violet (n=3). B. GDF15 expression was successfully inhibited after transfection of GDF15-specific shRNA plasmids (GDF15sh) in KB and OECM1 cell lines. After 48 h, cells were harvested for western blot analysis. C. Silencing GDF15 sensitized cells to irradiation in HNC cells. OECM1 and KB cells were transfected with GDF15sh or the vector plasmids and then subjected to irradiation (0 to 6 Gy). After 14 days, the cell colonies were determined (n=3). D. GDF15 had minimal effect on growth regulation in HNC cells. The OECM1, Detroit and KB cells were treated with or without rhGDF15 (20 ng/ml for 5 days). After 14 days, the cell colonies were determined (n=3). (*: p <0.05, n.s. : non-significance, t -test).

Article Snippet: For administration of GDF15, the recombinant human GDF15 protein (rhGDF15) (#957-GD, R&D Systems, Minneapolis, MN) was added to the cell culture medium at 20 ng/ml for 2 to 5 days.

Techniques: Irradiation, Staining, Expressing, Transfection, shRNA, Western Blot, Plasmid Preparation

Journal: Oncotarget

Article Title: GDF15 contributes to radioresistance and cancer stemness of head and neck cancer by regulating cellular reactive oxygen species via a SMAD-associated signaling pathway

doi: 10.18632/oncotarget.13649

Figure Lengend Snippet: A. Administration of rhGDF15 suppressed intracellular ROS level in HNC cells. KB and OECM1 cells were treated with 20 ng/ml of rhGDF15 for 5 days. After staining with DCF dye, the cells were subjected to flow cytometric analysis to determine the intracellular ROS levels. All values are presented as fluorescence intensity. B. GDF15 silencing increased ROS production in HNC cells. After transfection with GDF15sh or the vector plasmids, the KB and OECM1 cells were stained with DCF dye and subjected to flow cytometric analysis. C. GDF15 silencing increased ROS production in esophageal cancer cells. CE48T/VGH and CE81T/VGH cells were co-transfected with DsRed and GDF15sh/vector plasmids and then subjected to 4 Gy of irradiation. After staining with DCF dye, the cells were subjected to confocal microscopy. D. GDF15 silencing reduced mitochondrial membrane potential and led to apoptosis in HNC cells. KB or Detroit cells were transfected with the GDF15sh or the vector plasmids for 48 h. After incubating with MitoCapture reagents, the cells were subjected to flow cytometry analysis. The PE fluorescence fraction represents healthy cells, and the FITC fraction represents apoptotic cells.

Article Snippet: For administration of GDF15, the recombinant human GDF15 protein (rhGDF15) (#957-GD, R&D Systems, Minneapolis, MN) was added to the cell culture medium at 20 ng/ml for 2 to 5 days.

Techniques: Staining, Fluorescence, Transfection, Plasmid Preparation, Irradiation, Confocal Microscopy, Membrane, Flow Cytometry

Journal: Oncotarget

Article Title: GDF15 contributes to radioresistance and cancer stemness of head and neck cancer by regulating cellular reactive oxygen species via a SMAD-associated signaling pathway

doi: 10.18632/oncotarget.13649

Figure Lengend Snippet: A. Elevation of GDF15 mRNA expressions in CD44+ sorted cells compared to the CD44- cells in both KB and OECM1 cell lines, as determined by RT-PCR method (n=3). B. Elevation of GDF15 protein levels in the conditional medium of CD44+ sorted cells compared to the CD44- cells in both KB and OECM1 cell lines, as determined by ELISA method (n=3). C. Elevation of GDF15 mRNA expressions in ALDH1+ sorted cells compared to the ALDH1- cells in both KB and OECM1 cell lines, as determined by RT-PCR method (n=3). D. Elevation of CD44 mRNA expressions in ALDH1+ sorted cells compared to the ALDH1- cells in both KB and OECM1 cell lines, as determined by RT-PCR method (n=3). E. Elevation of CD44+ population in rhGDF15 treated cells compared to non-treated cells in both KB and Detroit cell lines, as determined by FACS method. F. Reduction of CD44+ population in GDF15sh transfected cells in both KB and Detroit cell lines, as determined by FACS method. G. Elevation of ALDH1+ population in rhGDF15 treated cells in both KB and Detroit cell lines, as determined by FACS method. H. Reduction of ALDH1+ population in GDF15sh transfected cells in both KB and Detroit cell lines, as determined by FACS method. I. Enhancement of spheroid cell formation in the GDF15+ sorted cells compared to GDF15- cells in both KB and OECM1 cell lines (n=3). J. Reduction of spheroid cell formation in GDF15sh transfected cells in both Detroit and KB cell lines (n=3). K. Increase of invasion ability in the rhGDF15 treated cells compared to non-treated cells (control) in both Detroit and KB cell lines (n=3). L. Increase of migration ability in the rhGDF15 treated cells compared to non-treated cells (control) in both Detroit and KB cell lines (n=3). (*: p <0.05, **: p <0.01, ***: p <0.001, n.s. : non-significance, t -test).

Article Snippet: For administration of GDF15, the recombinant human GDF15 protein (rhGDF15) (#957-GD, R&D Systems, Minneapolis, MN) was added to the cell culture medium at 20 ng/ml for 2 to 5 days.

Techniques: Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transfection, Control, Migration

Journal: Oncotarget

Article Title: GDF15 contributes to radioresistance and cancer stemness of head and neck cancer by regulating cellular reactive oxygen species via a SMAD-associated signaling pathway

doi: 10.18632/oncotarget.13649

Figure Lengend Snippet: A. Treatment of antioxidant agent led to radioresistance in HNC cells. After treatment of 10 μM N-acetylcysteine (NAC) for 48 h, Detroit or KB cells were subjected to serial dose of irradiation (0 to 6 Gy). Fourteen days later, the numbers of surviving cell colonies were determined (n=3). B. Treatment of antioxidant agent increase CD44+ cell population. After treatment of 10 μM NAC for 48 h, the OECM1 or KB cells were subjected to flow cytometry analysis for CD44+ populations. C. GDF15 reversed the effects of ROS in the suppression of spheroid cell formation. Detroit or KB cells were treated with H 2 O 2 (5 μM) with or without addition of rhGDF15 (20 ng/ml) for 48 h. These cells were then incubated in the spheroid cell culture condition and assessed after 14 days (n=3). (*: p < 0.05, **: p < 0.01, ***: p < 0.001, t -test).

Article Snippet: For administration of GDF15, the recombinant human GDF15 protein (rhGDF15) (#957-GD, R&D Systems, Minneapolis, MN) was added to the cell culture medium at 20 ng/ml for 2 to 5 days.

Techniques: Irradiation, Flow Cytometry, Incubation, Cell Culture

Journal: Oncotarget

Article Title: GDF15 contributes to radioresistance and cancer stemness of head and neck cancer by regulating cellular reactive oxygen species via a SMAD-associated signaling pathway

doi: 10.18632/oncotarget.13649

Figure Lengend Snippet: A. GDF15 expression was reduced after treatment of TGF-β inhibitor. KB and OECM1 cells were treated with serial doses of LY364947 (5 to 20 μM) for 24 h. Cellular protein was extracted and subjected to western blot analysis to assess GDF15 protein expression. B. Administration of rhGDF15 increased TGF-β downstream molecule PAI-1 luciferase reporter activity. KB or Detroit cells were transfected with luciferase reporter plasmid carrying PAI-1 gene, with or without addition of rhGDF15 (20 ng/ml). After 48 h, cells were harvested for measurements of luciferase activity (n=3). C. Silencing rhGDF15 suppressed PAI-1 luciferase reporter activity. KB or Detroit cells were transfected with luciferase reporter plasmid carrying PAI-1 gene, with or without co-transfection of GDF15sh plasmid. After 48 h, cells were harvested for measurements of luciferase activity (n=3). D. PAI-1 expression induced by TGF-β was suppressed following GDF15 silencing. KB or Detroit cells were transfected with luciferase reporter plasmid carrying PAI-1 gene, with or without addition of rhGDF15 (20 ng/ml), or with or without co-transfection of GDF15sh plasmid. After 48 h, cells were harvested for measurements of luciferase activity (n=3). (*: p < 0.05, **: p < 0.01, ***: p < 0.001, t -test).

Article Snippet: For administration of GDF15, the recombinant human GDF15 protein (rhGDF15) (#957-GD, R&D Systems, Minneapolis, MN) was added to the cell culture medium at 20 ng/ml for 2 to 5 days.

Techniques: Expressing, Western Blot, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Cotransfection

Journal: Oncotarget

Article Title: GDF15 contributes to radioresistance and cancer stemness of head and neck cancer by regulating cellular reactive oxygen species via a SMAD-associated signaling pathway

doi: 10.18632/oncotarget.13649

Figure Lengend Snippet: A, B. Administration of rhGDF15 increased the phosphorylated forms of SMAD family proteins in HNC cells. KB or FaDu cells were treated with serial doses of rhGDF15 (0-10 ng/ml) for 15 min (A) or 5 ng/ml rhGDF15 for various times (0-120 min) (n=3). (B) The cellular proteins were extracted and subjected to western blot analysis for SMAD family protein expressions (n=3). C. Effects of SMAD1-siRNA on the expressions of SMAD family proteins. After transfection of SMAD1-specific siRNA or the scramble oligonucleotides in HNC cells for 48h, cellular proteins were extracted for western blot analysis. GAPDH protein was used as an internal control (n=3). D. Silencing SMAD1 increased ROS level in HNC cells. KB or OECM1 cells were transfected SMAD1 specific siRNA or the scramble oligonucleotides for 48 h. After treating cells with 10 μM H 2 O 2 for 20 min, the Intracellular ROS levels were determined by DCF dye staining and analyzed with flow cytometry. E. Silencing SMAD1 suppressed spheroid cell formation in HNC cells. Fadu, OECM1 or KB cells were transfected SMAD1 specific siRNA or the scramble oligonucleotides for 48 h. These cells were then incubated in the spheroid cell culture condition and assessed after 14 days (n=3). F. The spheroid cell formation promoted by GDF15 was inhibited in SMAD knockdown HNC cells. KB or OECM1 cells were transfected SMAD1 specific siRNA or the scramble oligonucleotides for 48 h, with the addition of rhGDF15 (20 ng/ml). These cells were then incubated in the spheroid cell culture condition and assessed after 14 days (n=3). G. Effects of SMAD3-siRNA on the expressions of SMAD family proteins. After transfection of SMAD3-specific siRNA or the scramble oligonucleotides in HNC cells for 48h, cellular proteins were extracted for western blot analysis. GAPDH protein was used as an internal control (n=3). H. Silencing SMAD3 had no effect on ROS level in HNC cells. Fadu or OECM1 cells were transfected SMAD3-specific siRNA or the scramble oligonucleotides for 48 h. After treating cells with 10 μM H 2 O 2 for 20 min, the Intracellular ROS levels were determined by DCF dye staining and analyzed with flow cytometry. I. Silencing SMAD3 had no significant effect on spheroid cell formation in HNC cells. Fadu, OECM1 or Detroit cells were transfected SMAD3 specific siRNA or the scramble oligonucleotides for 48 h, with the addition of rhGDF15 (20 ng/ml). After 14 days of incubation in the spheroid cell culture condition, cells were assessed for spheroid formation (n=3). (*: p < 0.05, **: p < 0.01, ***: p < 0.001, n.s. : non-significance, t -test).

Article Snippet: For administration of GDF15, the recombinant human GDF15 protein (rhGDF15) (#957-GD, R&D Systems, Minneapolis, MN) was added to the cell culture medium at 20 ng/ml for 2 to 5 days.

Techniques: Western Blot, Transfection, Control, Staining, Flow Cytometry, Incubation, Cell Culture, Knockdown

Journal: Oncotarget

Article Title: GDF15 contributes to radioresistance and cancer stemness of head and neck cancer by regulating cellular reactive oxygen species via a SMAD-associated signaling pathway

doi: 10.18632/oncotarget.13649

Figure Lengend Snippet: A total of 4×10 6 KB cells, with or without pre-treatment with the rhGDF15 protein (20 ng/ml for 5 days), were subcutaneously injected into BALB/c mice (10 mice each group) in the upper portion of the hind limb. At day 14, each group was randomly divided into two groups (5 mice per group), with or without receiving 2 Gy of irradiation, followed by repeated irradiation of the same dose twice a week for a total of 8 Gy. A. Tumor volume was measured twice a week and calculated as (length x width x height) for 36 days. B-D. The tumors in the group of irradiation, either with or without pre-treatment of rhGDF15, were dissected. The protein expression levels of SMAD family molecules in the rhGDF15 treatment tumor group (B) or the control groups (C) were determined by using western blot analysis, and quantified the relative expression levels after normalized with GAPDH (D). E. The expression levels of ALDH1 and Nestin in tumor tissues were determined by using IHC analysis. Three tumor sections of IHC staining were shown for examples (*: p < 0.05, **: p < 0.01, ***: p < 0.001, t -test).

Article Snippet: For administration of GDF15, the recombinant human GDF15 protein (rhGDF15) (#957-GD, R&D Systems, Minneapolis, MN) was added to the cell culture medium at 20 ng/ml for 2 to 5 days.

Techniques: Injection, Irradiation, Expressing, Control, Western Blot, Immunohistochemistry

Journal: Oncotarget

Article Title: GDF15 contributes to radioresistance and cancer stemness of head and neck cancer by regulating cellular reactive oxygen species via a SMAD-associated signaling pathway

doi: 10.18632/oncotarget.13649

Figure Lengend Snippet: Diagram of the mechanism by which GDF15 contributes to radioresistance and cancer stemness through regulating ROS levels via a SMAD-associated pathway

Article Snippet: For administration of GDF15, the recombinant human GDF15 protein (rhGDF15) (#957-GD, R&D Systems, Minneapolis, MN) was added to the cell culture medium at 20 ng/ml for 2 to 5 days.

Techniques:

Journal: Journal of cachexia, sarcopenia and muscle

Article Title: GDNF family receptor alpha-like antagonist antibody alleviates chemotherapy-induced cachexia in melanoma-bearing mice.

doi: 10.1002/jcsm.13219

Figure Lengend Snippet: Figure 1 Anti-GFRAL antibodies were selected by solution-phase biopanning. (A) Strategy for anti-GFRAL antibody selection using a human combina- torial antibody phage library. Black dotted lines represent GFRAL-binding phage pool. (B) Polyclonal phage ELISA for phage pools from each round of biopanning. (C) Heavy-chain complementarity-determining region 3 (H-CDR3) amino acid sequences of the anti-GFRAL antibody clones. (D) Binding of the anti-GFRAL antibodies to both human and mouse GFRAL extracellular domains verified by ELISA. (E) Luciferase reporter assay of HEK293 cells transfected with human GFRAL, human RET, and SRE-luciferase genes for the selection of the most potent GFRAL antagonist antibody with inhibitory activity against GDF15-induced luminescent signal (n = 5). Data are presented as the mean ± standard error of the mean (SEM), analysed with the Kruskal–Wallis test, followed by the uncorrected Dunn’s test. Statistical differences in post hoc testing are indicated as ns = non-significant, ***P < 0.005.

Article Snippet: Following that, 5-fold serial dilutions of A11 starting at 20 μg/mL or unbiotinylated recombinant human GDF15 (8146-GD; R&D Systems) controls starting at 180 μM were added to plates for 1 h at 37°C.

Techniques: Selection, Binding Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Luciferase, Reporter Assay, Transfection, Activity Assay

Journal: Journal of cachexia, sarcopenia and muscle

Article Title: GDNF family receptor alpha-like antagonist antibody alleviates chemotherapy-induced cachexia in melanoma-bearing mice.

doi: 10.1002/jcsm.13219

Figure Lengend Snippet: Figure 2 The antibody A11 binds to GFRAL and inhibits the signal transduction of the GDF15/GFRAL/RET axis. (A) SPR sensorgram showing the binding kinetics of the antibody A11 to the recombinant human GFRAL extracellular domain. Black box indicates concentration of antibody A11 (1.89– 31.25 nM). (B) Flow cytometry analysis of the control antibody and antibody A11 binding in wild-type (WT) and human GFRAL gene-transfected HEK293 cells. Black box indicates concentration of antibody A11 (0–50 nM). (C) Representative immunostaining images of commercial anti-GFRAL an- tibody (left) and antibody A11 (right) binding in WT and human GFRAL gene-transfected HEK293 cells. The white scale bar represents 30 μm. (D) West- ern blot analysis showing the inhibitory effect of the antibody A11 on the phosphorylation of RET, AKT, and ERK in human GFRAL/RET gene-transfected HEK293 cells. The gel is representative of three independent experiments.

Article Snippet: Following that, 5-fold serial dilutions of A11 starting at 20 μg/mL or unbiotinylated recombinant human GDF15 (8146-GD; R&D Systems) controls starting at 180 μM were added to plates for 1 h at 37°C.

Techniques: Transduction, Binding Assay, Recombinant, Concentration Assay, Flow Cytometry, Control, Transfection, Immunostaining, Phospho-proteomics

Journal: Journal of cachexia, sarcopenia and muscle

Article Title: GDNF family receptor alpha-like antagonist antibody alleviates chemotherapy-induced cachexia in melanoma-bearing mice.

doi: 10.1002/jcsm.13219

Figure Lengend Snippet: Figure 3 The antibody A11 attenuates cisplatin-induced cachexia in vivo Normal: control mice with PBS injections, Cis/Con: mice with cisplatin and control antibody injections, Cis/A11: mice with cisplatin and the antibody A11 injections (n = 7 mice per group). (A) Cumulative food intake per mouse on days 2–5. (B) Body weight change compared with the weight before drug administration followed up to day 5. (C) Weights of isolated skeletal mus- cles (quadriceps, gastrocnemius, and soleus). (D) Weights of isolated adipose tissues (eWAT and iWAT). (E) Plasma levels of GDF15 on day 5, deter- mined by ELISA. Data are presented as the mean ± SEM, analysed with the Kruskal–Wallis test, followed by the uncorrected Dunn’s test. Statistical differences in post hoc testing are indicated as ns = non-significant, *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001.

Article Snippet: Following that, 5-fold serial dilutions of A11 starting at 20 μg/mL or unbiotinylated recombinant human GDF15 (8146-GD; R&D Systems) controls starting at 180 μM were added to plates for 1 h at 37°C.

Techniques: In Vivo, Control, Isolation, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Journal of cachexia, sarcopenia and muscle

Article Title: GDNF family receptor alpha-like antagonist antibody alleviates chemotherapy-induced cachexia in melanoma-bearing mice.

doi: 10.1002/jcsm.13219

Figure Lengend Snippet: Figure 4 The antibody A11 suppresses c-Fos accumulation on the hindbrain of cisplatin-treated mice Normal: control mice with PBS injections, Cis/ Con: mice with cisplatin and control antibody injections, Cis/A11: mice with cisplatin and the antibody A11 injections (n = 5 mice per group). (A) Rep- resentative sections of the area postrema (AP) and the nucleus of the solitary tract (NTS) showing c-Fos expression in GFRAL-expressing neurons 4 h after cisplatin treatment on day 4. White arrows represent neurons stained with both GFRAL and c-Fos, indicating activation by cisplatin-induced GDF15. The white scale bars represent 150 μm and the yellow scale bar represents 20 μm. The CC represents central canal. (B) Quantification of cisplatin-induced c-Fos expression in the AP/NTS 4 h after cisplatin injection. Each point represents one mouse. Data from each mouse represent quan- tification from 6 alternative sections. (C) Number of GFRAL-positive neurons in the AP/NTS that co-express c-Fos 4 h after cisplatin injection. Each point represents one mouse. Data from each mouse represent quantification from 6 alternative sections. Data are presented as the mean ± SEM, analysed with the Kruskal–Wallis test, followed by the uncorrected Dunn’s test. Statistical differences in post hoc testing are indicated as ns = non-significant, *P < 0.05, **P < 0.01.

Article Snippet: Following that, 5-fold serial dilutions of A11 starting at 20 μg/mL or unbiotinylated recombinant human GDF15 (8146-GD; R&D Systems) controls starting at 180 μM were added to plates for 1 h at 37°C.

Techniques: Control, Expressing, Staining, Activation Assay, Injection

Journal: Journal of cachexia, sarcopenia and muscle

Article Title: GDNF family receptor alpha-like antagonist antibody alleviates chemotherapy-induced cachexia in melanoma-bearing mice.

doi: 10.1002/jcsm.13219

Figure Lengend Snippet: Figure 5 The antibody A11 ameliorates cisplatin-induced cachexia in a mouse model of melanoma Normal: control mice without tumour, TB/Con: tu- mour-bearing mice with control antibody injections, TB/A11: tumour-bearing mice with the antibody A11 injections, TB/Cis/Con: tumour-bearing mice with cisplatin and control antibody injections, TB/Cis/A11: tumour-bearing mice with cisplatin and the antibody A11 injections (n = 10–16 mice per group). (A) Cumulative food intake per mouse on days 13–18. (B) Body weight change compared with the weight before drug administration followed up to day 17. (C) Weights of isolated skeletal muscles (quadriceps, gastrocnemius, and soleus). (D) Weights of isolated adipose tissues (eWAT and iWAT). (E) Plasma GDF15 levels on day 18, determined by ELISA. Data are presented as the mean ± SEM, analysed with the Kruskal–Wallis test, followed by the uncorrected Dunn’s test. Statistical differences in post hoc testing are indicated as ns = non-significant, *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001.

Article Snippet: Following that, 5-fold serial dilutions of A11 starting at 20 μg/mL or unbiotinylated recombinant human GDF15 (8146-GD; R&D Systems) controls starting at 180 μM were added to plates for 1 h at 37°C.

Techniques: Control, Isolation, Muscles, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Growth/differentiation factor 15 controls number of ependymal and neural stem cells in the ventricular/subventricular zone

doi: 10.1101/2022.12.02.518869

Figure Lengend Snippet: (A) Immunofluorescent staining of brain slices from 8w-old mice for GDF15 (green) and either Sox2 or EGFR (red). DAPI was used as nuclear counterstain. V= ventricle; scale bar = 20 μm. (B, C) Immunofluorescent staining of brain slices from E18 (B) or 8w-old mice (C) for GFRAL (green). DAPI was used as nuclear counterstain. V= ventricle; scale bar = 20 μm.

Article Snippet: For whole mount preparations, brain tissue of 8-weeks-old animals or E18 embryos was dissected as described before , and the GE or SVZ was directly fixed in a 3% PFA / 4% sucrose solution in PBS for 24 h. Alternatively, the tissue was incubated in a well of a 24-well-plate containing 1 ml Euromed-N (Euroclone) with 1x B27 supplement (Invitrogen) and as indicated either a solvent control, human recombinant GDF15 (10 ng/ml; R&D Systems, 9279-GD), human recombinant EGF (20 ng/ml; Peprotech, #AF-100-15), PD158780 (20 μM; Calbiochem/Merck, #513035) or AMD3100 octahydrochloride hydrate (6 μM; Sigma Aldrich, # A5602) at 37°C, 5% CO2 for 24h, and then fixed as described above.

Techniques: Staining

Journal: bioRxiv

Article Title: Growth/differentiation factor 15 controls number of ependymal and neural stem cells in the ventricular/subventricular zone

doi: 10.1101/2022.12.02.518869

Figure Lengend Snippet: (A) Percentage of Prominin-expressing cells in the E18 GE of wild-type (WT) and Gdf15 -/- mice analysed by flow cytometry. (B) Immunofluorescent staining of E18 GE whole mounts for proliferation marker Ki67 (green). Confocal images are taken at the apical side of the GE. DAPI was used as nuclear counterstain. White arrow indicates a mitotic cell. V= ventricle; scale bar = 20 μm. (C, D) Quantification of total number of Ki67 + cells (C) and mitotic cells in the apical and subapical cell layers (D) in E18 whole mounts. (E) Immunofluorescent staining of E18 GE whole mounts for proliferation marker phH3 (red) and IdU (green). Images are taken at the apical side of the GE. The dissected tissue was incubated in IdU for 1.5h and directly fixed, or further incubated without IdU for 12h. DAPI was used as nuclear counterstain. Scale bars = 20 μm. (F) Quantification of cells labelled with IdU and phH3 directly after IdU application (1.5h) or after a 12h chase period (12h). Cells expressing phH3 were counted separately depending on location of the nucleus (apical or subapical) to infer the type of progenitor. Bars represent mean ± SEM; * indicate significance: *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: For whole mount preparations, brain tissue of 8-weeks-old animals or E18 embryos was dissected as described before , and the GE or SVZ was directly fixed in a 3% PFA / 4% sucrose solution in PBS for 24 h. Alternatively, the tissue was incubated in a well of a 24-well-plate containing 1 ml Euromed-N (Euroclone) with 1x B27 supplement (Invitrogen) and as indicated either a solvent control, human recombinant GDF15 (10 ng/ml; R&D Systems, 9279-GD), human recombinant EGF (20 ng/ml; Peprotech, #AF-100-15), PD158780 (20 μM; Calbiochem/Merck, #513035) or AMD3100 octahydrochloride hydrate (6 μM; Sigma Aldrich, # A5602) at 37°C, 5% CO2 for 24h, and then fixed as described above.

Techniques: Expressing, Flow Cytometry, Staining, Marker, Incubation

Journal: bioRxiv

Article Title: Growth/differentiation factor 15 controls number of ependymal and neural stem cells in the ventricular/subventricular zone

doi: 10.1101/2022.12.02.518869

Figure Lengend Snippet: (A) Sketch illustrating the different division modes determined by the angle of the mitotic spindle relative to an orthogonal axis to the ventricular surface. Categories were determined as follows: 0° = 0°-30°; 45° = 30°-60°; 90° = 60°-90°. (B, C) Quantification of total number of mitotic cells according to angle of mitosis at E18 (B) and in adult animals (C). (D, E) Quantification of mitotic cells according to angle normalized to total mitotic cells at E18 (D) and in adult animals (E). (F, G) Quantification of Ki67 + cells (F) or mitotic cells (G) in E18 GE whole mounts either incubated without (Ctrl) or with GDF15 overnight. Bars represent mean ± SEM; */ + indicate significance: */ + p<0.05, **p<0.01, ***/ +++ p<0.001; * indicates significance to respective WT control, + indicates significance to respective untreated control.

Article Snippet: For whole mount preparations, brain tissue of 8-weeks-old animals or E18 embryos was dissected as described before , and the GE or SVZ was directly fixed in a 3% PFA / 4% sucrose solution in PBS for 24 h. Alternatively, the tissue was incubated in a well of a 24-well-plate containing 1 ml Euromed-N (Euroclone) with 1x B27 supplement (Invitrogen) and as indicated either a solvent control, human recombinant GDF15 (10 ng/ml; R&D Systems, 9279-GD), human recombinant EGF (20 ng/ml; Peprotech, #AF-100-15), PD158780 (20 μM; Calbiochem/Merck, #513035) or AMD3100 octahydrochloride hydrate (6 μM; Sigma Aldrich, # A5602) at 37°C, 5% CO2 for 24h, and then fixed as described above.

Techniques: Incubation, Control

Journal: bioRxiv

Article Title: Growth/differentiation factor 15 controls number of ependymal and neural stem cells in the ventricular/subventricular zone

doi: 10.1101/2022.12.02.518869

Figure Lengend Snippet: (A) Immunofluorescent staining for EGFR in slices from E18 mice. White arrows show the columns of radially oriented EGFR labelled cells that are absent in the Gdf15 -/- GE. V = ventricle, VZ = ventricular zone; scale bar = 50 μm. (B) Quantification of EGFR immunofluorescence intensity of the immunostaining shown in (A). (C-E) Fold change in EGFR-expressing (E + ) cells isolated from the WT (C) and Gdf15 -/- (D, E) GE at E18 as analysed by flow cytometry. Cells were either incubated in medium without or with GDF15, or with addition of CXCR4 antagonist AMD3100 (AMD), as indicated. Bars represent mean ± SEM; * indicates significance: *p<0.05, **p<0.01.

Article Snippet: For whole mount preparations, brain tissue of 8-weeks-old animals or E18 embryos was dissected as described before , and the GE or SVZ was directly fixed in a 3% PFA / 4% sucrose solution in PBS for 24 h. Alternatively, the tissue was incubated in a well of a 24-well-plate containing 1 ml Euromed-N (Euroclone) with 1x B27 supplement (Invitrogen) and as indicated either a solvent control, human recombinant GDF15 (10 ng/ml; R&D Systems, 9279-GD), human recombinant EGF (20 ng/ml; Peprotech, #AF-100-15), PD158780 (20 μM; Calbiochem/Merck, #513035) or AMD3100 octahydrochloride hydrate (6 μM; Sigma Aldrich, # A5602) at 37°C, 5% CO2 for 24h, and then fixed as described above.

Techniques: Staining, Immunofluorescence, Immunostaining, Expressing, Isolation, Flow Cytometry, Incubation

Journal: bioRxiv

Article Title: Growth/differentiation factor 15 controls number of ependymal and neural stem cells in the ventricular/subventricular zone

doi: 10.1101/2022.12.02.518869

Figure Lengend Snippet: (A) Coronal sections of the GE germinal region of E18 WT and Gdf15 -/- mice after immunostaining for phH3 (green) and EGFR (red). DAPI was used as nuclear counterstain. AB = apical border, VZ/SVZ = ventricular zone/subventricular zone, GE = deep ganglionic eminence, V = lateral ventricle. Scale bar = 37.5 μm. (B) Quantification of phH3 + cells co-expressing EGFR in the different parts of the niche. AB = Apical border. (C-E) Quantification of Ki67 + cells (C) or mitotic cells (D, E) at the apical side of E18 GE whole mounts either untreated (Ctrl) or incubated with EGFR-blocker PD158780 (PD) or exogenous EGF as indicated. (F) Percentage of Prominin + cells determined by FACS after overnight incubation with EGF. Data points represent individual animals, lines connect EGF-treated and untreated (Ctrl) data point of the for the same animal. Significance determined by paired t-test. (G-I) Quantification of Ki67 + cells (G) or mitotic cells (H, I) at the apical side of E18 GE whole mounts either incubated with or with DMSO as control or CXCR4-blocker AMD3100 overnight. (J) Clonal analysis of cells treated with AMD3100 after FACS sort. Bars represent mean ± SEM; */ + indicate significance: */ + p<0.05, **/ ++ p<0.01, ***/ +++ p<0.001; * indicates significance to respective WT control, + indicates significance to respective untreated control.

Article Snippet: For whole mount preparations, brain tissue of 8-weeks-old animals or E18 embryos was dissected as described before , and the GE or SVZ was directly fixed in a 3% PFA / 4% sucrose solution in PBS for 24 h. Alternatively, the tissue was incubated in a well of a 24-well-plate containing 1 ml Euromed-N (Euroclone) with 1x B27 supplement (Invitrogen) and as indicated either a solvent control, human recombinant GDF15 (10 ng/ml; R&D Systems, 9279-GD), human recombinant EGF (20 ng/ml; Peprotech, #AF-100-15), PD158780 (20 μM; Calbiochem/Merck, #513035) or AMD3100 octahydrochloride hydrate (6 μM; Sigma Aldrich, # A5602) at 37°C, 5% CO2 for 24h, and then fixed as described above.

Techniques: Immunostaining, Expressing, Incubation, Control

Journal: bioRxiv

Article Title: Growth/differentiation factor 15 controls number of ependymal and neural stem cells in the ventricular/subventricular zone

doi: 10.1101/2022.12.02.518869

Figure Lengend Snippet: (A) BrdU + cells (green) and DAPI counterstaining of the nuclei (blue) in WT and Gdf15 -/- control neurosphere cultures fixed two days after plating (DAP) in differentiation medium. BrdU was added to the culture medium 24h before fixation. Scale bar = 20 μm. (B, C) Quantitative analysis of BrdU + cells fixed two days (B) or seven days after induction of differentiation (C), with recombinant GDF15 added during differentiation (+GDF15) or without (Ctrl). (D) Representative examples of neurosphere cultures derived from the E18 GE fixed seven days after the induction of differentiation and stained for TuJ1. DAPI was used for nuclear counterstain. Scale bar = 20 μm. (E, F) Quantitative analyses of the number of TuJ1 + neurons in cultures fixed seven (E) and ten (F) days after the induction of differentiation and treated with exogenous GDF15 during the entire phase of differentiation (GDF15) or left untreated (Control) as indicated. Bars represent mean ± SEM; * indicates significance: *p<0.05.

Article Snippet: For whole mount preparations, brain tissue of 8-weeks-old animals or E18 embryos was dissected as described before , and the GE or SVZ was directly fixed in a 3% PFA / 4% sucrose solution in PBS for 24 h. Alternatively, the tissue was incubated in a well of a 24-well-plate containing 1 ml Euromed-N (Euroclone) with 1x B27 supplement (Invitrogen) and as indicated either a solvent control, human recombinant GDF15 (10 ng/ml; R&D Systems, 9279-GD), human recombinant EGF (20 ng/ml; Peprotech, #AF-100-15), PD158780 (20 μM; Calbiochem/Merck, #513035) or AMD3100 octahydrochloride hydrate (6 μM; Sigma Aldrich, # A5602) at 37°C, 5% CO2 for 24h, and then fixed as described above.

Techniques: Control, Recombinant, Derivative Assay, Staining

Journal: bioRxiv

Article Title: Growth/differentiation factor 15 controls number of ependymal and neural stem cells in the ventricular/subventricular zone

doi: 10.1101/2022.12.02.518869

Figure Lengend Snippet: (A) Quantitative analysis of the percentage of DAPI stained nuclei displaying BrdU immunoreactivity per region of interest (ROI) within the GE of E18 GE from WT and Gdf15 -/- embryos 2 and 6 hours after BrdU injection. (B) Coronal sections of the V/SVZ of adult WT and Gdf15 -/- mice after immunostaining for Ki67 (green) and Mash1 (red). DAPI was used as nuclear counterstain. Scale bar = 20 μm. (C, D) Quantitative analysis of the percentage of DAPI stained nuclei in the V/SVZ of the adult WT and Gdf15 -/- animals immunoreactive to MASH1 and displaying Ki67 immunoreactivity as indicated. Lat. wall = lateral wall, dors. corner = dorsolateral corner. Bars represent mean ± SEM; * indicates significance: *p<0.05, **p<0.01.

Article Snippet: For whole mount preparations, brain tissue of 8-weeks-old animals or E18 embryos was dissected as described before , and the GE or SVZ was directly fixed in a 3% PFA / 4% sucrose solution in PBS for 24 h. Alternatively, the tissue was incubated in a well of a 24-well-plate containing 1 ml Euromed-N (Euroclone) with 1x B27 supplement (Invitrogen) and as indicated either a solvent control, human recombinant GDF15 (10 ng/ml; R&D Systems, 9279-GD), human recombinant EGF (20 ng/ml; Peprotech, #AF-100-15), PD158780 (20 μM; Calbiochem/Merck, #513035) or AMD3100 octahydrochloride hydrate (6 μM; Sigma Aldrich, # A5602) at 37°C, 5% CO2 for 24h, and then fixed as described above.

Techniques: Staining, Injection, Immunostaining

Journal: Cell Research

Article Title: Neurotensin-neurotensin receptor 2 signaling in adipocytes suppresses food intake through regulating ceramide metabolism

doi: 10.1038/s41422-024-01038-8

Figure Lengend Snippet: a Expression levels of Gdf15 in the adipose tissues ( n = 4–7). b – d GDF15 protein concentrations in the serum of control and Ntsr2 AKO mice fed by a chow diet ( b , n = 8–11), HFD ( c , n = 8–12) or treated by NTS ( d , n = 5). e Illustration of the experimental design. f Food intake of control and Ntsr2 AKO mice with or without knockdown of Gfral ( n = 8). g Food intake of mice treated by NTS in iWATs with Gfral knockdown ( n = 5). * P < 0.05; *** P < 0.001; ns, not significant.

Article Snippet: The levels of GDF15 protein in the serum were measured by an ELISA kit (Cat# MGD150, R&D Systems) following the manufacturer’s instructions.

Techniques: Expressing, Control, Knockdown

Journal: Cell Research

Article Title: Neurotensin-neurotensin receptor 2 signaling in adipocytes suppresses food intake through regulating ceramide metabolism

doi: 10.1038/s41422-024-01038-8

Figure Lengend Snippet: a Expression level of Gdf15 upon NTS treatment in the primary adipocytes of WT mice ( n = 3–4). b – d Expression levels of GDF15 protein upon CerS2 knockdown ( b , n = 3–5), CerS2 overexpression ( c , n = 6) and ceramide C22 treatment ( d , n = 3–4) in primary adipocytes. e – g Serum concentrations of GDF15 ( e , n = 6), mRNA expression levels of Gdf15 ( f , n = 3) and GDF15 protein abundance in adipose tissues ( g , n = 3) of control and CerS2 +/– mice. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The levels of GDF15 protein in the serum were measured by an ELISA kit (Cat# MGD150, R&D Systems) following the manufacturer’s instructions.

Techniques: Expressing, Knockdown, Over Expression, Quantitative Proteomics, Control