Journal: Materials Today Bio

Article Title: Dual-functional ROS-responsive hydrogel alleviates temporomandibular joint osteoarthritis by enhancing cartilage repair and mitigating inflammation

doi: 10.1016/j.mtbio.2025.102103

Figure Lengend Snippet: Schematic illustration of the mechanism by which ROS-sensitive THG hydrogel encapsulating FGF18 active protein (FGF18/THG) treats temporomandibular joint (TMJ) osteoarthritis (TMJOA). A ROS-responsive, biodegradable anti-inflammatory hydrogel (THG) is designed by combining HA–GA with N1-(4-boronobenzyl)-N3-(4-boronophenyl)-N1, N1, N3, N3-tetramethylpropane-1,3-diaminium (TSPBA). The hydrogel encapsulates FGF18 (FGF18/THG), which is injected into the TMJ cavity. Upon exposure to the reactive oxygen species (ROS)-enriched TMJOA microenvironment, the boronic ester groups in FGF18/THG degrade, leading to ROS depletion. Simultaneously, the released FGF18 and anti-inflammatory molecules (e.g., GA) further neutralize ROS and inhibit the abnormally activated NF-κB pathway in osteoarthritic condylar chondrocytes, upregulating cartilage-specific genes ( Col2 , Acan ) and downregulating pro-inflammatory genes (e.g., Adamts5, Mmp13 ). This synergistic process promotes cartilage regeneration, reduces inflammation, and prevents extracellular matrix (ECM) degradation. Additionally, in milder cases of TMJOA, the therapeutic effects of FGF18 are further enhanced, slowing disease progression in vivo .

Article Snippet: The resulting solutions and FGF18 (HY-P7124, MCE, New Jersey, NJ, USA) were stored at −20 °C for later use.

Techniques: Injection, Biomarker Discovery, In Vivo

Journal: Materials Today Bio

Article Title: Dual-functional ROS-responsive hydrogel alleviates temporomandibular joint osteoarthritis by enhancing cartilage repair and mitigating inflammation

doi: 10.1016/j.mtbio.2025.102103

Figure Lengend Snippet: Characterization and properties of THG hydrogel. (A) Schematic of hyaluronic acid (HA)-gallic acid (GA) polymer synthesis, a precursor of THG hydrogel. (B) Schematic of N1-(4-boronobenzyl)-N3-(4-boronophenyl)-N1, N1, N3, N3-tetramethylpropane-1,3-diaminium (TSPBA) polymer synthesis, a precursor of THG hydrogel. (C) 1 H NMR spectra of HA, HA–NH 2 , and HA–GA in D 2 O. (D) 1 H NMR spectrum of TSPBA. (E) Schematic illustration of gel formation: HA–GA mixed with TSPBA at a 2:1 vol ratio forms an opaque white gel. (F) Scanning electron microscopy shows the formation of a porous network in THG hydrogel (scale bars: 50 μm and 10 μm). (G) Rheological evaluation of the storage (G′) and loss (G″) moduli of THG hydrogel at 37 °C. (H) Release profile of FGF18 from reactive oxygen species (ROS)-sensitive THG hydrogels immersed in 10 μM H 2 O 2 or phosphate-buffered saline (PBS) solutions. (I) Degradation pattern of ROS-sensitive THG hydrogels immersed in 10 μM H 2 O 2 or PBS solutions. Values are presented as the mean ± standard error of the mean (SEM) of three independent experiments. Significance was determined by Tukey's multiple comparison test following two-way analysis of variance (ANOVA). ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: The resulting solutions and FGF18 (HY-P7124, MCE, New Jersey, NJ, USA) were stored at −20 °C for later use.

Techniques: Polymer, Electron Microscopy, Saline, Comparison

Journal: Materials Today Bio

Article Title: Dual-functional ROS-responsive hydrogel alleviates temporomandibular joint osteoarthritis by enhancing cartilage repair and mitigating inflammation

doi: 10.1016/j.mtbio.2025.102103

Figure Lengend Snippet: In vitro anti-inflammatory and cytotoxicity evaluation of FGF18/THG. (A) Representative fluorescence images of the reactive oxygen species (ROS) levels in condylar chondrocytes co-cultured with FGF18, THG, and FGF18/THG using ROS fluorescence staining. (B) Immunofluorescence staining of catalase (CAT) and superoxide dismutase 1 (SOD1) expression in IL-1β-induced condylar chondrocytes treated with FGF18/THG. (C) Quantitative evaluation of ROS levels in condylar chondrocytes using ROS fluorescence staining. (D) mRNA expression of Cat and Sod1 in IL-1β-induced condylar chondrocytes treated with FGF18/THG. (E) CCK-8 assay results showing the cytotoxicity of FGF18/THG on condylar chondrocytes based on cell proliferation. Values are presented as the mean ± standard error of the mean (SEM) of three independent experiments. Significance was determined by one-way analysis of variance (ANOVA): ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: The resulting solutions and FGF18 (HY-P7124, MCE, New Jersey, NJ, USA) were stored at −20 °C for later use.

Techniques: In Vitro, Fluorescence, Cell Culture, Staining, Immunofluorescence, Expressing, CCK-8 Assay

Journal: Materials Today Bio

Article Title: Dual-functional ROS-responsive hydrogel alleviates temporomandibular joint osteoarthritis by enhancing cartilage repair and mitigating inflammation

doi: 10.1016/j.mtbio.2025.102103

Figure Lengend Snippet: Effect of FGF18/THG on cartilage matrix synthesis and degradation in IL-1β-induced inflamed condylar chondrocytes. (A) RT-qPCR analysis of relative mRNA expression levels of Col2 , Acan , Adamts5 , and Mmp13 in IL-1β-induced condylar chondrocytes co-cultured with FGF18/THG. (B) Western blot detection of COL2, ACAN, ADAMTS5, and MMP13 proteins in IL-1β-induced inflamed condylar chondrocytes co-cultured with FGF18/THG. (C) Semi-quantitative analysis of the immunoblotting results. Values are presented as the mean ± standard error of the mean (SEM) of three independent experiments. Significance was determined by one-way analysis of variance (ANOVA): ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, ∗∗∗∗ P < 0.0001. The cells were primary condylar chondrocytes isolated from Sprague–Dawley (SD) rats.

Article Snippet: The resulting solutions and FGF18 (HY-P7124, MCE, New Jersey, NJ, USA) were stored at −20 °C for later use.

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Western Blot, Isolation

Journal: Materials Today Bio

Article Title: Dual-functional ROS-responsive hydrogel alleviates temporomandibular joint osteoarthritis by enhancing cartilage repair and mitigating inflammation

doi: 10.1016/j.mtbio.2025.102103

Figure Lengend Snippet: Macroscopic evaluation and histopathological assessment of the temporomandibular joint (TMJ) in SD rats with experimental TMJ osteoarthritis (TMJOA) after two weeks of intra-joint injection treatment with FGF18/THG. (A) Schematic diagram of the SD rat TMJOA experimental model, indicating the time points for partial discectomy, intra-joint injection of FGF18, THG, or FGF18/THG, and sacrifice. (B) Representative macroscopic images of the TMJ condyles in sham-operated and PDE-operated rats after eight weeks of treatment with FGF18/HA–GA or control. (C) Representative histological images stained with H&E and Safranin O/fast green showing histological changes in cartilage sections from different groups of SD rats eight weeks post-PDE surgery. (D) Representative immunohistochemical images of COL2, ACAN, ADAMTS5, and MMP13 staining in the TMJ condyles of rats two weeks post-sham surgery or PDE surgery, treated with FGF18/THG or control. The dashed box highlights the repair area, and arrows indicate the inflammatory regions. (E) Macroscopic semi-quantitative scoring of the TMJ condylar cartilage in SD rats. (F) Semi-quantitative scoring of Safranin O/fast green staining in the TMJ condylar cartilage of different groups of SD rats. (G) Semi-quantitative analysis of COL2, ACAN, ADAMTS5, and MMP13 immunohistochemical staining in TMJ cartilage of different groups of SD rats. Values are presented as the mean ± standard error of the mean (SEM) of four independent experiments. Significance was determined by one-way analysis of variance (ANOVA): ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: The resulting solutions and FGF18 (HY-P7124, MCE, New Jersey, NJ, USA) were stored at −20 °C for later use.

Techniques: Injection, Control, Staining, Immunohistochemical staining

Journal: Materials Today Bio

Article Title: Dual-functional ROS-responsive hydrogel alleviates temporomandibular joint osteoarthritis by enhancing cartilage repair and mitigating inflammation

doi: 10.1016/j.mtbio.2025.102103

Figure Lengend Snippet: FGF18/HA–GA treats temporomandibular joint osteoarthritis (TMJOA) by inhibiting the NF-κB pathway. (A) Representative p-IKK immunohistochemical staining images of temporomandibular joint (TMJ) condyles from SD rats treated with FGF18/HA–GA or control, eight weeks post-sham surgery and partial discectomy (PDE) surgery. (B) Semi-quantitative analysis of p-IKK immunohistochemical staining in TMJ cartilage of different groups of SD rats. (C) Western blot analysis of p-IKK and p-p65 protein expression levels in IL-1β-induced inflamed condylar chondrocytes co-cultured with FGF18/THG. (D) Semi-quantitative analysis of the immunoblotting results for each group. Values are presented as the mean ± standard error of the mean (SEM) of three independent experiments. (E) Relative mRNA expression levels of Tnfa and Il6 in IL-1β-induced condylar chondrocytes co-cultured with FGF18/THG. (F) Protein expression of p-p65, TNF-α, and IL-6 in IL-1β-induced condylar chondrocytes co-cultured with FGF18/THG. Significance was determined by one-way analysis of variance (ANOVA): ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: The resulting solutions and FGF18 (HY-P7124, MCE, New Jersey, NJ, USA) were stored at −20 °C for later use.

Techniques: Immunohistochemical staining, Staining, Control, Western Blot, Expressing, Cell Culture

Journal: Nature Communications

Article Title: Transgenerational inheritance of diminished ovarian reserve triggered by prenatal propylparaben exposure in mice

doi: 10.1038/s41467-025-63440-z

Figure Lengend Snippet: a STRING database revealed strong interactions between RhoBTB1 and FGF18, RhoBTB1 and CTSK, respectively. b , c The protein levels of FGF18 and MAPK signaling pathway in KGN cells detected by western blotting following PrP exposure. Data from ( c ) are representative of three independent experiments (mean ± SEM; one-way ANOVA). d , e The protein levels of FGF18 and MAPK signaling pathway in KGN cells detected by western blotting following RhoBTB1 over-expression. Data from ( e ) are representative of three independent experiments (mean ± SEM; unpaired two-tailed t -test). f , g The protein levels of FGF18, RhoBTB1, and MAPK signaling pathway in GCs detected by western blotting following PrP exposure and siRNA-F02 interference of Fgf18 . Data from ( g ) are representative of three independent experiments (mean ± SEM; one-way ANOVA). h , i The protein levels of RhoBTB1, FGF18, and MAPK signaling pathway in GCs detected by western blotting following PrP exposure and siRNA-R03 interference of Rhobtb1 . Data from ( i ) are representative of three independent experiments (mean ± SEM; one-way ANOVA). j , k The protein levels of FGF18 in GCs following PrP exposure and MLN4924 treatment. Data from ( k ) are representative of three independent experiments (mean ± SEM; one-way ANOVA). l , m The protein levels of FGF18 in GCs following RhoBTB1 over-expression and MLN4924 treatment. Data from ( m ) are representative of three independent experiments (mean ± SEM; unpaired two-tailed t -test). n , o The protein levels of FGF18 in GCs following PrP exposure and MG132 treatment. Data from ( o ) are representative of three independent experiments (mean ± SEM; one-way ANOVA). p , q The protein levels of FGF18 in GCs following RhoBTB1 over-expression and MG132 treatment. Data from ( q ) are representative of three independent experiments (mean ± SEM; unpaired two-tailed t -test). The samples derived from the same experiment and that blots were processed in parallel. Source data are provided as a Source Data file.

Article Snippet: The Antibodies were purchased from different companies: Cell Signaling Technology for ERK (Extracellular Signal-Regulated Kinase; 4695T), p-ERK (Phosphorylated ERK; 4370T), p38 (p38 Mitogen-Activated Protein Kinase; 8690T), p-p38 (Phosphorylated p38; 4511T), JNK (c-Jun N-terminal Kinase; 9252T), and p-JNK (Phosphorylated JNK; 4668T); Abclonal for β-ACTIN (AC038), active-Caspase3 (A19654), BCL-2 (A19693), BAX (A12009), SOD2 (A19576), BMP15 (A7321), CAT (A11777), HSD17B7 (A17157), and CTSK (A1782); Proteintech for FGF18 (60341-1-lg); Immunoway for RhoBTB1 (YT4084); Servicebio for HRP conjugated goat anti-rabbit IgG (GB23303), and Alexa Fluor® 488-conjugated goat anti-rabbit IgG (GB25303).

Techniques: Western Blot, Over Expression, Two Tailed Test, Derivative Assay

Journal: Biochemical and biophysical research communications

Article Title: A high-sensitivity ELISA for detection of human FGF18 in culture supernatants from tumor cell lines.

doi: 10.1016/j.bbrc.2023.06.070

Figure Lengend Snippet: Fig. 1. Generation of anti-human FGF18 antibodies. a, Alignment of the consensus motif recognized by furin (RXXR) and the C-terminal amino acids of hFGF18. The RXXR motif is underlined, and the potential cleavage sites are shown as vertical arrows. b, Reactivity of 6 rabbit monoclonal antibodies to hFGF18. Purified human FGF18DC (hFGF18DC) from HEK293 cells was serially diluted and coated onto a plate. The coated hFGF18DC8 was detected with 1 mg/mL biotinylated 6 rabbit monoclonal antibodies followed by incubation with streptavidin-HRP. c, Comparison of the binding activities of clones 12S-SE5 and 12G7-9 and a commercially available rabbit polyclonal antibody (PA120525) to hFGF18. Purified hFGF18DC was coated and detected with 1 mg/mL of the indicated antibodies as described in b. d, Clone 19S-SE5 less efficiently binds to mouse FGF18 (mFGF18). Recombinant full-length mFGF18 was serially diluted and coated onto a plate. The coated proteins were detected as in b. Results are represen- tative of two independent experiments.

Article Snippet: We coated serially diluted purified hFGF18DC and detected them using clones 19S-SE5 and 12G7-9 and a commercially available rabbit polyclonal anti-hFGF18 antibody (Cusabio CSBPA120525).

Techniques: Bioprocessing, Incubation, Comparison, Binding Assay, Clone Assay, Recombinant

Journal: Biochemical and biophysical research communications

Article Title: A high-sensitivity ELISA for detection of human FGF18 in culture supernatants from tumor cell lines.

doi: 10.1016/j.bbrc.2023.06.070

Figure Lengend Snippet: Fig. 2. 19S-SE5 specifically detects the C-terminal region of hFGF18. a, Alignment of human and murine FGF18 at protein levels. The first 26 amino acids indicate the signal peptides. DC8 and DC25 indicate the deleted amino acids from the C-terminus. The boxes indicate different amino acids between human and mouse. b, Coomassie blue staining of hFGF18DC8 and hFGF18DC25. GST-hFGF18DC25 was generated and purified in E. coli as described in the Materials and Methods section. Five micrograms of hFGF18DC8 and hFGF18DC25 were subjected to SDS‒PAGE and stained with Coomassie blue. c, d, Deletion of the last 25 amino acids completely and partially abolishes the binding activities of clones 19S-SE5 (c) and 12G7-9 (d), respectively. Purified hFGF18DC8 and hFGF18DC25 were serially diluted and coated onto the plate. The signals were detected as in Fig. 1b. Results are representative of two independent experiments.

Article Snippet: We coated serially diluted purified hFGF18DC and detected them using clones 19S-SE5 and 12G7-9 and a commercially available rabbit polyclonal anti-hFGF18 antibody (Cusabio CSBPA120525).

Techniques: Staining, Generated, Binding Assay, Clone Assay

Journal: Biochemical and biophysical research communications

Article Title: A high-sensitivity ELISA for detection of human FGF18 in culture supernatants from tumor cell lines.

doi: 10.1016/j.bbrc.2023.06.070

Figure Lengend Snippet: Fig. 4. Comparison of the sensitivity of the in-house ELISA to detect hFGF18 and mFGF18. a, Coomassie blue staining of full-length mFGF18 and mFGF18DC8. GST-mFGF18 and GST- mFGF18DC8 were generated in E. coli and purified as in Fig. 2b. After digestion with PreScission protease, 5 mg of GST, mFGF18, and mFGF18DC8 were subjected to SDS‒PAGE and stained with Coomassie blue. b, Comparison of the sensitivity of the in-house ELISA to full-length mFGF18 and mFGF18DC8. The serially diluted proteins were coated, and the concentrations of the indicated proteins were determined by the in-house ELISA. c, The in-house ELISA detected mFGF18DC8, albeit the sensitivity is relatively low compared to hFGF18DC8. The serially diluted hFGF18DC8 and mFGF18DC8 were coated, and the concentrations of hFGF18DC8 and mFGF18D8 were determined by the in-house ELISA. The right panel is the enlarged image of the dotted box in the left panel. Results are representative of two independent experiments. d, mFGF18 is elevated in sera from Fgf18 Tg mice. Sera from 6- to 8-week-old Non-Tg and Fgf18 Tg mice and concentrations of mFGF18 were determined by the in-house ELISA. Results are the mean ± SE (n ¼ 5 mice). Statistical sig- nificance was determined using a two-tailed unpaired Student's t-test. Results were pooled from two independent experiments.

Article Snippet: We coated serially diluted purified hFGF18DC and detected them using clones 19S-SE5 and 12G7-9 and a commercially available rabbit polyclonal anti-hFGF18 antibody (Cusabio CSBPA120525).

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Staining, Generated, Two Tailed Test

Journal: Nature Communications

Article Title: FGF18 alleviates hepatic ischemia-reperfusion injury via the USP16-mediated KEAP1/Nrf2 signaling pathway in male mice

doi: 10.1038/s41467-023-41800-x

Figure Lengend Snippet: A RNA sequence was carried out between I/R and Sham. The results of FGFs changes are presented in the form of heat maps ( n = 3). B The protein expression of FGF18 in human subjected to hepatic resection ( n = 4). C The protein expression of FGF18 in livers of mice subjected to I/R (1 h/6 h) surgery or not ( n = 3). Non-ischemia represented non-ischemia liver lobes and ischemia represented ischemia liver lobes. D The ratio of FGF18/GAPDH after hepatectomy positively correlated positive with ALT at POD1 ( n = 32). E The serum level of FGF18 in human subjected to hepatic resection ( n = 5). F The protein expression of FGF18 in different tissues under hepatic I/R (1 h/6 h) or not ( n = 3). G KCs, LSECs, HSCs, and hepatocytes were extracted from mouse liver and subjected to H/R (4 h/6 h) challenge. All the primary liver cells were verified for purity by immunofluorescence. Scale bar = 200 μm. Then cells were carried out for RT-PCR assay ( n = 3). (Scheme is Created with BioRender.com) H Immunofluorescence for FGF18 (red) and different cell markers (green). Scale bar = 50 μm ( n = 5). I/R ischemia reperfusion, HSCs hepatic stellate cells, KC Kupffer cell, LSEC liver sinusoids endothelial cell; The statistical significance of differences were assessed by two-tailed student unpaired t -test for ( B , C , E ). Other assays were assessed by one-way ANOVA, followed by Tukey’s multiple comparison test. Data are presented as means ± SEM with individual values. All numbers ( n ) are biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: ELISA assays were performed to evaluate the serum FGF18 levels in human (CUSABIO, K05010174) and mice (JINGMEI BIOTECHNOLOGY, JM-12187M1) according to the manufacturer’s instructions.

Techniques: Sequencing, Expressing, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: FGF18 alleviates hepatic ischemia-reperfusion injury via the USP16-mediated KEAP1/Nrf2 signaling pathway in male mice

doi: 10.1038/s41467-023-41800-x

Figure Lengend Snippet: Mice were injected with AAV2/9-FGF18 via tail tein. After stably expressed for two weeks, mice were subjected to I/R (1 h/6 h) surgery. A Schematic diagram of the work and western blotting showed the transfection efficiency of AAV-FGF18 ( n = 5). (Scheme is Created with BioRender.com) B Serum ALT and AST level ( n = 5). C H&E staining of liver sections. Scale bar=100 μm ( n = 5). D – F TUNEL staining, c-CAS-3 immunohistochemistry, and MPO staining of liver sections ( n = 5). Scale bar = 100 μm. G Relative protein expression by western blotting ( n = 3). H Protein levels of apoptosis related signaling pathway in H/R (4 h/6 h) treated L02 cells in the presence of FGF18 (200 ng/mL) subjected to challenge or not ( n = 3). I , J Relative mRNA expression of genes by RT-PCR ( n = 3). H/R, hypoxia/reoxygenation; The statistical significance of differences were assessed by two-tailed two-tailed student unpaired t -test for C – F . Other assays were assessed by one-way ANOVA, followed by Tukey’s multiple comparison test. Data are presented as means ± SEM with individual values. All numbers (n) are biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: ELISA assays were performed to evaluate the serum FGF18 levels in human (CUSABIO, K05010174) and mice (JINGMEI BIOTECHNOLOGY, JM-12187M1) according to the manufacturer’s instructions.

Techniques: Injection, Stable Transfection, Western Blot, Transfection, Staining, TUNEL Assay, Immunohistochemistry, Expressing, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: FGF18 alleviates hepatic ischemia-reperfusion injury via the USP16-mediated KEAP1/Nrf2 signaling pathway in male mice

doi: 10.1038/s41467-023-41800-x

Figure Lengend Snippet: Fgf18 △HSC mice were constructed by using Fgf18 f/f and GFAP-specific Cre mice. A Schematic diagram of mouse breeding. And the genotype of Fgf18 △HSC mice and the knockdown efficiency of FGF18 were verified by RT-PCR ( n = 3). (Scheme is Created with BioRender.com). B Serum ALT and AST level ( n = 5). C H&E staining of liver sections ( n = 5). Scale bar = 100 μm. D – F TUNEL staining, c-CAS-3 immunohistochemistry, and MPO staining of liver sections ( n = 5). Scale bar=100 μm. G Relative protein expression by western blotting ( n = 3). H RNA-seq analysis of livers from Fgf18 f/f mice and Fgf18 △HSC mice subjected to I/R (1 h/6 h). Red indicated upregulation while blue indicated downregulation ( n = 3). The statistical significance of differences were assessed by two-tailed student unpaired t -test for ( C – F ). Other assays were assessed by one-way ANOVA, followed by Tukey’s multiple comparison test. Data are presented as means ± SEM with individual values. All numbers ( n ) are biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: ELISA assays were performed to evaluate the serum FGF18 levels in human (CUSABIO, K05010174) and mice (JINGMEI BIOTECHNOLOGY, JM-12187M1) according to the manufacturer’s instructions.

Techniques: Construct, Knockdown, Reverse Transcription Polymerase Chain Reaction, Staining, TUNEL Assay, Immunohistochemistry, Expressing, Western Blot, RNA Sequencing, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: FGF18 alleviates hepatic ischemia-reperfusion injury via the USP16-mediated KEAP1/Nrf2 signaling pathway in male mice

doi: 10.1038/s41467-023-41800-x

Figure Lengend Snippet: A Protein level of KEAP1 was analyzed in HepG2 cells treated with FGF18 or not under H/R (4 h/6 h) challenge. The above cells were simultaneously subjected to MG132 (10 μM) or CQ (50 μM) in the process of H/R (4 h/6 h) ( n = 3). B Relative mRNA expression of genes by RT-PCR ( n = 3). C The peptide sequences of the KEAP1 protein was detected by mass spectrometry. D Immunofluorescence for KEAP1 (red) and USP16 (green). Scale bar = 20 μm. The white line represented the statistical position ( n = 5). E PLA assays ( n = 5). Scale bar = 50 μm. F Representative Co-immunoprecipitation experiment of KEAP1 and USP16 in HEK293T cells ( n = 3). G Co-immunoprecipitation experiment was carried out to determine the specific binding domain between Flag-USP16 and HA-KEAP1 ( n = 3). H Relative protein expression by western blotting ( n = 3). I In vitro GST pull-down assay analysis of the interaction of USP16 and KEAP1 ( n = 3). J HepG2 cells transfected with si- USP16 were treated with CHX, and the cells were collected at the indicated times ( n = 3). The statistical significance of differences were assessed by one-way ANOVA, followed by Tukey’s multiple comparison test. Data are presented as means ± SEM with individual values. All numbers ( n ) are biologically independent experiments. Source data are provided as a Source Data file. (n.s. not significant.).

Article Snippet: ELISA assays were performed to evaluate the serum FGF18 levels in human (CUSABIO, K05010174) and mice (JINGMEI BIOTECHNOLOGY, JM-12187M1) according to the manufacturer’s instructions.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Mass Spectrometry, Immunofluorescence, Immunoprecipitation, Binding Assay, Western Blot, In Vitro, Pull Down Assay, Transfection, Comparison

Journal: Nature Communications

Article Title: FGF18 alleviates hepatic ischemia-reperfusion injury via the USP16-mediated KEAP1/Nrf2 signaling pathway in male mice

doi: 10.1038/s41467-023-41800-x

Figure Lengend Snippet: A The interaction of KEAP1 and USP16 in HepG2 was detected in H/R (4 h/6 h) challenge or not ( n = 3). B The ubiquitination level of KEAP1 was determined in the indicated group in HepG2 cells ( n = 3). C The ubiquitination level of KEAP1 was determined in the indicated group in HEK293 cells ( n = 3). D HA-Ub or HA-Ub (K48-Mut) was transfected into HepG2 cells in the presence of si- USP16 or si- scramble . The ubiquitination level of KEAP1 was detected in the indicated group ( n = 3). E Flag-USP16 overexpressed HepG2 were transfected with HA-KEAP1 (WT) or HA-KEAP1 (domain Mut). Relative protein expression by western blotting. F The ubiquitination level of KEAP1 was determined in the indicated group in HepG2 cells ( n = 3). G HepG2 cells were subjected to H/R (4 h/6 h) challenge in the presence of FGF18. Then cells were simultaneously transfected Flag-USP16, Flag-USP16 C205S , or not. Relative protein expression by western blotting ( n = 3). The statistical significance of differences were assessed by two-tailed student unpaired t -test for ( E ). Other assays were assessed by one-way ANOVA, followed by Tukey’s multiple comparison test. Data are presented as means ± SEM with individual values. All numbers ( n ) are biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: ELISA assays were performed to evaluate the serum FGF18 levels in human (CUSABIO, K05010174) and mice (JINGMEI BIOTECHNOLOGY, JM-12187M1) according to the manufacturer’s instructions.

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Western Blot, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: FGF18 alleviates hepatic ischemia-reperfusion injury via the USP16-mediated KEAP1/Nrf2 signaling pathway in male mice

doi: 10.1038/s41467-023-41800-x

Figure Lengend Snippet: Fgf18 △HSC mice were injected with AAV8-TBG-sh- USP16 via tail tein. After stably expressed for two weeks, mice were subjected to I/R (1 h/6 h) surgery. A Schematic diagram of the work ( n = 5) (Scheme is Created with BioRender.com). B Serum ALT and AST level ( n = 5). C H&E staining of liver sections. Scale bar = 100 μm. D – G TUNEL staining, c-CAS-3 immunohistochemistry, MPO, and 4-HNE staining of liver sections ( n = 5). Scale bar = 100 μm. H Relative protein expression by western blotting ( n = 3). I Liver GSH and MDA levels ( n = 5). The statistical significance of differences were assessed by two-tailed student unpaired t -test. Data are presented as means ± SEM with individual values. All numbers ( n ) are biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: ELISA assays were performed to evaluate the serum FGF18 levels in human (CUSABIO, K05010174) and mice (JINGMEI BIOTECHNOLOGY, JM-12187M1) according to the manufacturer’s instructions.

Techniques: Injection, Stable Transfection, Staining, TUNEL Assay, Immunohistochemistry, Expressing, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: FGF18 alleviates hepatic ischemia-reperfusion injury via the USP16-mediated KEAP1/Nrf2 signaling pathway in male mice

doi: 10.1038/s41467-023-41800-x

Figure Lengend Snippet: A Protein levels of USP16 and HO-1 in H/R (4 h/6 h) treated HepG2 cells in the presence or absence of FGF18 or ML385 (10 μM), or not ( n = 3 per group) ( n = 3). B Relative mRNA expression of genes by RT-PCR ( n = 3). C Relative protein expression by western blotting ( n = 3). D Relative mRNA expression of genes by RT-PCR ( n = 3). E Relative luciferase activity was detected ( n = 3). F Binding of Nrf2 to the USP16 promoter region in H/R treated HepG2 cells was examined by CHIP assay ( n = 3). The statistical significance of differences were assessed by two-tailed student unpaired t -test for ( C , D ). Other assays were assessed by one-way ANOVA, followed by Tukey’s multiple comparison test. Data are presented as means ± SEM with individual values. All numbers ( n ) are biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: ELISA assays were performed to evaluate the serum FGF18 levels in human (CUSABIO, K05010174) and mice (JINGMEI BIOTECHNOLOGY, JM-12187M1) according to the manufacturer’s instructions.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Luciferase, Activity Assay, Binding Assay, Two Tailed Test, Comparison

Journal: Journal of Clinical Investigation

Article Title: Ubiquitin ligase RNF146 coordinates bone dynamics and energy metabolism

doi: 10.1172/jci92233

Figure Lengend Snippet: Figure 4. RNF146 regulates osteoblast differentiation through the FGF18-TAZ axis. (A and B) qPCR analysis of Fgf18 mRNA expression in cells in Figure 3I (A) or C (B), cultured in osteogenic medium for 1–3 days. (C) Luciferase activity from an osteocalcin reporter assay in C2C12 cells transfected with or without RUNX2, and cultured in serum-free medium in the presence or absence of FGF18 (50 ng/ml). (D) Western blot analysis in primary murine osteoblasts cul- tured in serum-free medium in the presence or absence of FGF18 (50 ng/ml). (E) qPCR analysis of Taz mRNA expression in cells in D. (F) Luciferase activity from an osteocalcin reporter assay in C2C12 cells infected with shGFP or shTaz, transfected with or without RUNX2 and cultured in serum-free medium in the presence or absence of FGF18 (50 ng/ml). (G) Western blot analysis in primary murine osteoblasts infected with shGFP or shFgf18 and cultured in serum- free medium in the presence or absence of WNT3a (40 ng/ml). (H) Cells in Figure 3C, infected with an empty vector control (mock) or an FGF18-expressing retroviral vector were cultured in osteogenic medium and stained with alizarin red S solution. (I) qPCR analysis of Taz mRNA expression in primary murine osteoblasts cultured in serum-free medium in the presence or absence of FGF18 (50 ng/ml) and U0126 (10 μM). (J) Western blot analysis in cells in I cultured for 10 minutes or 24 hours. (K) Luciferase activity from an osteocalcin reporter assay in C2C12 cells transfected with or without RUNX2 and cultured in serum-free medium in the presence or absence of FGF18 (50 ng/ml) and U0126 (10 μM). n = 3. P values were determined by ANOVA with Tukey–Kramer’s post-hoc test (A, C, F, G, and I–K) or unpaired t test (B, D, and E). Data are presented as mean ± SEM. *P < 0.05.

Article Snippet: FGF18 cDNA was purchased from Origene Technologies, and pMXs-FGF18 vector was constructed as described previously (40).

Techniques: Expressing, Cell Culture, Luciferase, Activity Assay, Reporter Assay, Transfection, Western Blot, Infection, Plasmid Preparation, Control, Retroviral, Staining

Journal: Journal of Clinical Investigation

Article Title: Ubiquitin ligase RNF146 coordinates bone dynamics and energy metabolism

doi: 10.1172/jci92233

Figure Lengend Snippet: Figure 5. RNF146 is required for osteoblast proliferation. (A) Growth curves of Rnf146fl/fl mouse–derived calvarial osteoblasts infected with GFP- or Cre- expressing adenovirus and cultured in growth medium for 2 days. (B) qPCR analysis of Ctgf mRNA expression in C2C12 cells infected with shGFP or shTaz and cultured in serum-free medium in the presence or absence of FGF18 (50 ng/ml). (C) Growth curves of C2C12 cells infected with shGFP or shTaz and cultured in serum-free medium in the presence or absence of FGF18 (50 ng/ml) for 2 days. (D) Bright-field images of Rnf146fl/fl mouse–derived calvarial osteoblasts infected with GFP- or Cre-expressing adenovirus and cultured in growth medium in the presence or absence of FGF18 (50 ng/ml) for 2 days. Original magnification: ×200. (E) Growth curves of cells in D cultured for 2 days. (F–M) qPCR analysis of Rnf146 (F), Axin1 (G), cyclin D1 (H), Fgf18 (I), Taz (J), Runx2 (K), Osx (L), and osteocalcin (M) mRNA expression in the calvarium derived from Rnf146fl/fl and Rnf146fl/fl Osx-Cre newborn pups. n = 3. P values were determined by ANOVA with Tukey-Kramer’s post-hoc test (B, C, and E) or unpaired t test (A and F–M). Data are presented as mean ± SEM. *P < 0.05.

Article Snippet: FGF18 cDNA was purchased from Origene Technologies, and pMXs-FGF18 vector was constructed as described previously (40).

Techniques: Derivative Assay, Infection, Expressing, Cell Culture

Journal: Nature Communications

Article Title: FGF18 alleviates hepatic ischemia-reperfusion injury via the USP16-mediated KEAP1/Nrf2 signaling pathway in male mice

doi: 10.1038/s41467-023-41800-x

Figure Lengend Snippet: A RNA sequence was carried out between I/R and Sham. The results of FGFs changes are presented in the form of heat maps ( n = 3). B The protein expression of FGF18 in human subjected to hepatic resection ( n = 4). C The protein expression of FGF18 in livers of mice subjected to I/R (1 h/6 h) surgery or not ( n = 3). Non-ischemia represented non-ischemia liver lobes and ischemia represented ischemia liver lobes. D The ratio of FGF18/GAPDH after hepatectomy positively correlated positive with ALT at POD1 ( n = 32). E The serum level of FGF18 in human subjected to hepatic resection ( n = 5). F The protein expression of FGF18 in different tissues under hepatic I/R (1 h/6 h) or not ( n = 3). G KCs, LSECs, HSCs, and hepatocytes were extracted from mouse liver and subjected to H/R (4 h/6 h) challenge. All the primary liver cells were verified for purity by immunofluorescence. Scale bar = 200 μm. Then cells were carried out for RT-PCR assay ( n = 3). (Scheme is Created with BioRender.com) H Immunofluorescence for FGF18 (red) and different cell markers (green). Scale bar = 50 μm ( n = 5). I/R ischemia reperfusion, HSCs hepatic stellate cells, KC Kupffer cell, LSEC liver sinusoids endothelial cell; The statistical significance of differences were assessed by two-tailed student unpaired t -test for ( B , C , E ). Other assays were assessed by one-way ANOVA, followed by Tukey’s multiple comparison test. Data are presented as means ± SEM with individual values. All numbers ( n ) are biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: LX-2 cells were transfected with si- FGF18 (Santa Cruz, sc-39478) by using Lipofectamine 2000 for 6 h in Opti-MEM.

Techniques: Sequencing, Expressing, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: FGF18 alleviates hepatic ischemia-reperfusion injury via the USP16-mediated KEAP1/Nrf2 signaling pathway in male mice

doi: 10.1038/s41467-023-41800-x

Figure Lengend Snippet: Mice were injected with AAV2/9-FGF18 via tail tein. After stably expressed for two weeks, mice were subjected to I/R (1 h/6 h) surgery. A Schematic diagram of the work and western blotting showed the transfection efficiency of AAV-FGF18 ( n = 5). (Scheme is Created with BioRender.com) B Serum ALT and AST level ( n = 5). C H&E staining of liver sections. Scale bar=100 μm ( n = 5). D – F TUNEL staining, c-CAS-3 immunohistochemistry, and MPO staining of liver sections ( n = 5). Scale bar = 100 μm. G Relative protein expression by western blotting ( n = 3). H Protein levels of apoptosis related signaling pathway in H/R (4 h/6 h) treated L02 cells in the presence of FGF18 (200 ng/mL) subjected to challenge or not ( n = 3). I , J Relative mRNA expression of genes by RT-PCR ( n = 3). H/R, hypoxia/reoxygenation; The statistical significance of differences were assessed by two-tailed two-tailed student unpaired t -test for C – F . Other assays were assessed by one-way ANOVA, followed by Tukey’s multiple comparison test. Data are presented as means ± SEM with individual values. All numbers (n) are biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: LX-2 cells were transfected with si- FGF18 (Santa Cruz, sc-39478) by using Lipofectamine 2000 for 6 h in Opti-MEM.

Techniques: Injection, Stable Transfection, Western Blot, Transfection, Staining, TUNEL Assay, Immunohistochemistry, Expressing, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: FGF18 alleviates hepatic ischemia-reperfusion injury via the USP16-mediated KEAP1/Nrf2 signaling pathway in male mice

doi: 10.1038/s41467-023-41800-x

Figure Lengend Snippet: Fgf18 △HSC mice were constructed by using Fgf18 f/f and GFAP-specific Cre mice. A Schematic diagram of mouse breeding. And the genotype of Fgf18 △HSC mice and the knockdown efficiency of FGF18 were verified by RT-PCR ( n = 3). (Scheme is Created with BioRender.com). B Serum ALT and AST level ( n = 5). C H&E staining of liver sections ( n = 5). Scale bar = 100 μm. D – F TUNEL staining, c-CAS-3 immunohistochemistry, and MPO staining of liver sections ( n = 5). Scale bar=100 μm. G Relative protein expression by western blotting ( n = 3). H RNA-seq analysis of livers from Fgf18 f/f mice and Fgf18 △HSC mice subjected to I/R (1 h/6 h). Red indicated upregulation while blue indicated downregulation ( n = 3). The statistical significance of differences were assessed by two-tailed student unpaired t -test for ( C – F ). Other assays were assessed by one-way ANOVA, followed by Tukey’s multiple comparison test. Data are presented as means ± SEM with individual values. All numbers ( n ) are biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: LX-2 cells were transfected with si- FGF18 (Santa Cruz, sc-39478) by using Lipofectamine 2000 for 6 h in Opti-MEM.

Techniques: Construct, Knockdown, Reverse Transcription Polymerase Chain Reaction, Staining, TUNEL Assay, Immunohistochemistry, Expressing, Western Blot, RNA Sequencing, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: FGF18 alleviates hepatic ischemia-reperfusion injury via the USP16-mediated KEAP1/Nrf2 signaling pathway in male mice

doi: 10.1038/s41467-023-41800-x

Figure Lengend Snippet: A Protein level of KEAP1 was analyzed in HepG2 cells treated with FGF18 or not under H/R (4 h/6 h) challenge. The above cells were simultaneously subjected to MG132 (10 μM) or CQ (50 μM) in the process of H/R (4 h/6 h) ( n = 3). B Relative mRNA expression of genes by RT-PCR ( n = 3). C The peptide sequences of the KEAP1 protein was detected by mass spectrometry. D Immunofluorescence for KEAP1 (red) and USP16 (green). Scale bar = 20 μm. The white line represented the statistical position ( n = 5). E PLA assays ( n = 5). Scale bar = 50 μm. F Representative Co-immunoprecipitation experiment of KEAP1 and USP16 in HEK293T cells ( n = 3). G Co-immunoprecipitation experiment was carried out to determine the specific binding domain between Flag-USP16 and HA-KEAP1 ( n = 3). H Relative protein expression by western blotting ( n = 3). I In vitro GST pull-down assay analysis of the interaction of USP16 and KEAP1 ( n = 3). J HepG2 cells transfected with si- USP16 were treated with CHX, and the cells were collected at the indicated times ( n = 3). The statistical significance of differences were assessed by one-way ANOVA, followed by Tukey’s multiple comparison test. Data are presented as means ± SEM with individual values. All numbers ( n ) are biologically independent experiments. Source data are provided as a Source Data file. (n.s. not significant.).

Article Snippet: LX-2 cells were transfected with si- FGF18 (Santa Cruz, sc-39478) by using Lipofectamine 2000 for 6 h in Opti-MEM.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Mass Spectrometry, Immunofluorescence, Immunoprecipitation, Binding Assay, Western Blot, In Vitro, Pull Down Assay, Transfection, Comparison

Journal: Nature Communications

Article Title: FGF18 alleviates hepatic ischemia-reperfusion injury via the USP16-mediated KEAP1/Nrf2 signaling pathway in male mice

doi: 10.1038/s41467-023-41800-x

Figure Lengend Snippet: A The interaction of KEAP1 and USP16 in HepG2 was detected in H/R (4 h/6 h) challenge or not ( n = 3). B The ubiquitination level of KEAP1 was determined in the indicated group in HepG2 cells ( n = 3). C The ubiquitination level of KEAP1 was determined in the indicated group in HEK293 cells ( n = 3). D HA-Ub or HA-Ub (K48-Mut) was transfected into HepG2 cells in the presence of si- USP16 or si- scramble . The ubiquitination level of KEAP1 was detected in the indicated group ( n = 3). E Flag-USP16 overexpressed HepG2 were transfected with HA-KEAP1 (WT) or HA-KEAP1 (domain Mut). Relative protein expression by western blotting. F The ubiquitination level of KEAP1 was determined in the indicated group in HepG2 cells ( n = 3). G HepG2 cells were subjected to H/R (4 h/6 h) challenge in the presence of FGF18. Then cells were simultaneously transfected Flag-USP16, Flag-USP16 C205S , or not. Relative protein expression by western blotting ( n = 3). The statistical significance of differences were assessed by two-tailed student unpaired t -test for ( E ). Other assays were assessed by one-way ANOVA, followed by Tukey’s multiple comparison test. Data are presented as means ± SEM with individual values. All numbers ( n ) are biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: LX-2 cells were transfected with si- FGF18 (Santa Cruz, sc-39478) by using Lipofectamine 2000 for 6 h in Opti-MEM.

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Western Blot, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: FGF18 alleviates hepatic ischemia-reperfusion injury via the USP16-mediated KEAP1/Nrf2 signaling pathway in male mice

doi: 10.1038/s41467-023-41800-x

Figure Lengend Snippet: Fgf18 △HSC mice were injected with AAV8-TBG-sh- USP16 via tail tein. After stably expressed for two weeks, mice were subjected to I/R (1 h/6 h) surgery. A Schematic diagram of the work ( n = 5) (Scheme is Created with BioRender.com). B Serum ALT and AST level ( n = 5). C H&E staining of liver sections. Scale bar = 100 μm. D – G TUNEL staining, c-CAS-3 immunohistochemistry, MPO, and 4-HNE staining of liver sections ( n = 5). Scale bar = 100 μm. H Relative protein expression by western blotting ( n = 3). I Liver GSH and MDA levels ( n = 5). The statistical significance of differences were assessed by two-tailed student unpaired t -test. Data are presented as means ± SEM with individual values. All numbers ( n ) are biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: LX-2 cells were transfected with si- FGF18 (Santa Cruz, sc-39478) by using Lipofectamine 2000 for 6 h in Opti-MEM.

Techniques: Injection, Stable Transfection, Staining, TUNEL Assay, Immunohistochemistry, Expressing, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: FGF18 alleviates hepatic ischemia-reperfusion injury via the USP16-mediated KEAP1/Nrf2 signaling pathway in male mice

doi: 10.1038/s41467-023-41800-x

Figure Lengend Snippet: A Protein levels of USP16 and HO-1 in H/R (4 h/6 h) treated HepG2 cells in the presence or absence of FGF18 or ML385 (10 μM), or not ( n = 3 per group) ( n = 3). B Relative mRNA expression of genes by RT-PCR ( n = 3). C Relative protein expression by western blotting ( n = 3). D Relative mRNA expression of genes by RT-PCR ( n = 3). E Relative luciferase activity was detected ( n = 3). F Binding of Nrf2 to the USP16 promoter region in H/R treated HepG2 cells was examined by CHIP assay ( n = 3). The statistical significance of differences were assessed by two-tailed student unpaired t -test for ( C , D ). Other assays were assessed by one-way ANOVA, followed by Tukey’s multiple comparison test. Data are presented as means ± SEM with individual values. All numbers ( n ) are biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: LX-2 cells were transfected with si- FGF18 (Santa Cruz, sc-39478) by using Lipofectamine 2000 for 6 h in Opti-MEM.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Luciferase, Activity Assay, Binding Assay, Two Tailed Test, Comparison

Journal: Biology of reproduction

Article Title: Expression and function of fibroblast growth factor 18 in the ovarian follicle in cattle.

doi: 10.1095/biolreprod.110.084277

Figure Lengend Snippet: FIG. 1. Localization of FGF18 mRNA and its protein in the bovine ovary. A) Real-time PCR of FGF18 mRNA in theca cells, granulosa cells, and oocytes of abattoir origin. Steady-state mRNA levels were expressed relative to a theca cell sample with the DDCt method. Follicles were classified as healthy (H, n¼15), transitional (T, n¼21), or atretic (A, n¼7) based on E2:P ratios. Oocyte samples were three independent pools of .40 oocytes that gave robust housekeeping gene amplification. Immu- nohistochemical staining was observed in granulosa (g) and theca (t) cells of follicles (B) and in corpora lutea (cl; D). Weak background staining only was observed in stroma (s) or in the presence of excess blocking peptide (C and E). Photomicrographs were taken at3200 magnification; bars¼50 lm (B–E). Vertical bars in panel A without common letters are significantly different (P , 0.05); nd ¼ not detected.

Article Snippet: After blocking for 1 h in 0.2% Tween-Tris-buffered saline (TBS), blots were incubated with 1:2000 mouse anti-human FGF18 (H00008817-B01P; Novus Biologicals, Inc., Littleton, CO) for 2 h with agitation, followed by three washes with 0.2% Tween-TBS.

Techniques: Real-time Polymerase Chain Reaction, Amplification, Staining, Blocking Assay

Journal: Biology of reproduction

Article Title: Expression and function of fibroblast growth factor 18 in the ovarian follicle in cattle.

doi: 10.1095/biolreprod.110.084277

Figure Lengend Snippet: FIG. 2. Regulation of FGF18 mRNA abundance in granulosa and theca cells in vitro. Granulosa and theca cells from 2- to 5-mm follicles were cultured under serum-free conditions for 6 days with LH (theca cells only) or with FSH or IGF1 (granulosa cells only) (see Materials and Methods for details). Steady-state mRNA levels were measured by real-time PCR and expressed relative to a calibrator sample with the DDCt method. Data are means 6 SEM of three independent cultures, and bars with no common letters are significantly different (P , 0.05); nd ¼ not detectable.

Article Snippet: After blocking for 1 h in 0.2% Tween-Tris-buffered saline (TBS), blots were incubated with 1:2000 mouse anti-human FGF18 (H00008817-B01P; Novus Biologicals, Inc., Littleton, CO) for 2 h with agitation, followed by three washes with 0.2% Tween-TBS.

Techniques: In Vitro, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Biology of reproduction

Article Title: Expression and function of fibroblast growth factor 18 in the ovarian follicle in cattle.

doi: 10.1095/biolreprod.110.084277

Figure Lengend Snippet: FIG. 3. Effect of FGF18 on secretion of estradiol from bovine granulosa cells and on steady-state levels of mRNA encoding proteins critical for estradiol biosynthesis (upper row) and on progesterone secretion and concentration of mRNA-encoding proteins involved in progesterone biosynthesis (lower row). Granulosa cells from 2- to 5-mm follicles were cultured in serum-free medium containing FSH (10 ng/ml) for 6 days and treated with the stated doses of FGF18 for the last 4 days of culture. Steady-state mRNA levels were measured by real-time PCR and expressed relative to a calibrator sample with the DDCt method. Data are means 6 SEM of three independent cultures, and bars with no common letters are significantly different (P , 0.05).

Article Snippet: After blocking for 1 h in 0.2% Tween-Tris-buffered saline (TBS), blots were incubated with 1:2000 mouse anti-human FGF18 (H00008817-B01P; Novus Biologicals, Inc., Littleton, CO) for 2 h with agitation, followed by three washes with 0.2% Tween-TBS.

Techniques: Concentration Assay, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Biology of reproduction

Article Title: Expression and function of fibroblast growth factor 18 in the ovarian follicle in cattle.

doi: 10.1095/biolreprod.110.084277

Figure Lengend Snippet: FIG. 4. Effect of FGF18 on secretion of SERPINE2 protein and abundance of SERPINE2 mRNA in granulosa cell culture. Granulosa cells from 2- to 5-mm follicles were cultured in serum-free medium containing FSH (10 ng/ml) for 6 days and treated with the stated doses of FGF18 for the last 4 days of culture. Steady-state mRNA levels were measured by real-time PCR and expressed relative to a calibrator sample with the DDCt method. Protein secretion was measured by a Western blot of the culture medium, and blots of two independent cultures are shown with the samples in the same order as in the graph. Data are means 6 SEM of three independent cultures, and bars with no common letters are significantly different (P , 0.05).

Article Snippet: After blocking for 1 h in 0.2% Tween-Tris-buffered saline (TBS), blots were incubated with 1:2000 mouse anti-human FGF18 (H00008817-B01P; Novus Biologicals, Inc., Littleton, CO) for 2 h with agitation, followed by three washes with 0.2% Tween-TBS.

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Western Blot

Journal: Biology of reproduction

Article Title: Expression and function of fibroblast growth factor 18 in the ovarian follicle in cattle.

doi: 10.1095/biolreprod.110.084277

Figure Lengend Snippet: FIG. 5. FGF18 mRNA and protein abun- dance in granulosa cells and theca cells in healthy dominant (DF) and in subordinate (SF) follicles during emergence of a follic- ular wave. Follicles were collected from five cows when the follicles were of similar diameter (A) but differed in estradiol con- centration in the follicular fluid (B) and CYP19 mRNA abundance in granulosa cells (C). D) Steady-state FGF18 mRNA levels were measured by real-time PCR and expressed relative to a calibrator sample with the DDCt method. E) FGF18 protein levels in follicular fluid were measured by Western blot (blots are shown for four animals). Data are means 6 SEM, and asterisks denote significant differences be- tween DF and SF (*P , 0.05, **P , 0.01).

Article Snippet: After blocking for 1 h in 0.2% Tween-Tris-buffered saline (TBS), blots were incubated with 1:2000 mouse anti-human FGF18 (H00008817-B01P; Novus Biologicals, Inc., Littleton, CO) for 2 h with agitation, followed by three washes with 0.2% Tween-TBS.

Techniques: Real-time Polymerase Chain Reaction, Western Blot

Journal: Biology of reproduction

Article Title: Expression and function of fibroblast growth factor 18 in the ovarian follicle in cattle.

doi: 10.1095/biolreprod.110.084277

Figure Lengend Snippet: FIG. 6. Effect of FGF18 on proliferation and the cell cycle. Granulosa cells were cultured in serum-free medium containing FSH (10 ng/ml) for 6 days and treated with FGF18 for the last 4 days of culture. A) The proportion of cells in sub-G1 (light gray bars), G0/1 (white bars), S (dark gray bars), and G2/M (black bars) phases where mea- sured by flow cytometry. B) DNA fragmen- tation by gel electrophoresis. C) Proliferation measured by the MTT assay. D) Relative abundance of mRNA encoding four cell proliferation and death genes was measured by real-time PCR. Data are means 6 SEM of three independent cultures, and bars with no common letters are signifi- cantly different (P , 0.05). Asterisks identify cell-cycle stages significantly affected by FGF18 (A).

Article Snippet: After blocking for 1 h in 0.2% Tween-Tris-buffered saline (TBS), blots were incubated with 1:2000 mouse anti-human FGF18 (H00008817-B01P; Novus Biologicals, Inc., Littleton, CO) for 2 h with agitation, followed by three washes with 0.2% Tween-TBS.

Techniques: Cell Culture, Flow Cytometry, Nucleic Acid Electrophoresis, MTT Assay, Real-time Polymerase Chain Reaction