Journal: Theranostics

Article Title: Construction of human pluripotent stem cell-derived testicular organoids and their use as humanized testis models for evaluating the effects of semaglutide

doi: 10.7150/thno.104523

Figure Lengend Snippet: Conversion of hiPSCs into precursor testis-like cells by stepwise addition of small molecules. (A) Schematic illustration of the strategy to convert hiPSCs into precursor testis-like cells by using small molecules. (B-D) qRT-PCR data of relative gene expression after 4 and 7 days of monolayer differentiation for (B) lineage markers ( OCT4 and SOX2 , pluripotency; TBXT , PS; LHX1 and PAX2 , IM), (C) bipotential gonad markers ( WT1 , GATA4 , LHX9 , NR0B1 , HSD3B2 , GADD45G , ZFPM2 , and EMX2 ), and (D) Testis markers ( AMH , DHH , SOX9 , FGF9 , CLDN11 , HSD3B1 , CYP17A1 , and HSD17B3 ). The mRNA copy number of each gene was normalized with GAPDH . Gene expression was quantified relative to day 0 hiPSCs (mean ± SD, n = 3 independent experiments). (E-J) Immunofluorescence analysis was performed at days 0, 4, and 7 of monolayer differentiation. Bipotential gonad cells: GATA4. Sertoli cells: SOX9 and ZO-1. Leydig cells: STAR and HSD3B1. Basement membrane: Collagen IV. Nuclei were stained with DAPI (blue). Scale bar, 20 µm.

Article Snippet: Subsequently, in the next 3 days, 200 ng/mL FGF9 (Cat# HY-P73053, MedChemExpress), 10 ng/mL BMP4 (Cat# HY-P7007, MedChemExpress), and 1 μg/mL heparin (Cat# S1346, Selleckchem) were added to the differentiation medium.

Techniques: Quantitative RT-PCR, Gene Expression, Immunofluorescence, Membrane, Staining

Journal: Theranostics

Article Title: Construction of human pluripotent stem cell-derived testicular organoids and their use as humanized testis models for evaluating the effects of semaglutide

doi: 10.7150/thno.104523

Figure Lengend Snippet: hiPSC-derived pre-testis cells self-assembled into 3D cell spheroids in an environment that combined hanging drop and rotation culture. (A) Schematic illustration of the strategy for testicular organoid preparation. The dissociated day 7 monolayer cells were reaggregated and cultured in a hanging drop combined rotation system. (B-C) Bright-field images and area statistics of cell spheroids at densities of 1000, 3000, and 5000 cells/drop after 1, 3, and 5 days of hanging drop culture. Image scale bar, 100 µm. The area of cell spheroids was measured by ImageJ (mean ± SD, n = 50). (D) Bright-field images and Live/Dead staining images of cell spheroids (5000 cells/drop) after 3, 8, 13, and 18 days of organoid culture. Image scale bar, 200 µm. (E) The area of cell spheroids (5000 cells/drop) was measured by ImageJ (mean ± SD, n = 50). (F-I) qRT-PCR data of relative gene expression in day 0 and day 7 monolayer cells and day 3, 8, 13, and 18 organoids for (F) lineage markers ( OCT4 , pluripotency; PAX2 , IM), (G) bipotential gonad markers ( WT1 , GATA4 , NR0B1 , HSD3B2 , ZFPM2 , and EMX2 ), (H) Sertoli cell markers ( SOX9 , FGF9 , CLDN11 , and FSHR ), and (I) Leydig cell markers ( HSD3B1 , CYP17A1 , and HSD17B3 ). The mRNA copy number of each gene was normalized with GAPDH . Gene expression was quantified relative to day 0 hiPSCs (mean ± SD, n = 3 independent experiments). (J-M) Immunofluorescence analysis was conducted on days 3, 8, 13, and 18 organoids. Bipotential gonad cells were identified by the marker GATA4, Sertoli cells by SOX9, Leydig cells by HSD3B1, and peritubular myoid cells by α-SMA. Nuclei were counterstained with DAPI (blue). The image to the right of each merged image shows an enlargement of the white dashed square. Scale bars = 100 µm, and 50 µm in magnified regions.

Article Snippet: Subsequently, in the next 3 days, 200 ng/mL FGF9 (Cat# HY-P73053, MedChemExpress), 10 ng/mL BMP4 (Cat# HY-P7007, MedChemExpress), and 1 μg/mL heparin (Cat# S1346, Selleckchem) were added to the differentiation medium.

Techniques: Derivative Assay, Cell Culture, Staining, Quantitative RT-PCR, Gene Expression, Immunofluorescence, Marker

Journal: Theranostics

Article Title: Construction of human pluripotent stem cell-derived testicular organoids and their use as humanized testis models for evaluating the effects of semaglutide

doi: 10.7150/thno.104523

Figure Lengend Snippet: Combining network pharmacology and bioinformatics to identify reprogramming mechanisms during testicular organoid induction. (A) Schematic diagram of the initial hiPSCs-directed differentiation protocol for inducing testicular organoids. GFs: growth factors. (B) Heatmap of microarray data of hiPSC, day 3 organoids and day 8 organoids (|log 2 FoldChange| ≥ 2, Q value ≤ 0.01). Blue indicates decreased expression, and red indicates increased expression. (C) Number of up- or down-regulated genes between day 3 testicular organoids and hiPSC (|log 2 FoldChange| ≥ 2, Q value ≤ 0.01). (D) Volcano plot of the DEGs between day 3 organoids and hiPSC (|log 2 FoldChange| ≥ 2, Q value ≤ 0.01; up-regulation: red; down-regulation: blue). (E) The Venn diagram analysis of the DEGs (day 3 organoids vs hiPSC) and the predicted targets of reprogramming compounds (CHIR, Activin A, BMP4, FGF9 and heparin). (F) The network of the relationship between the reprogramming compounds and 109 common targets. Yellow diamond nodes represent targets, and square nodes represent reprogramming compounds. Lines represent interactions between compounds and targets; the size and color of reprogramming compound nodes are proportional to the number of associated targets. (G) The PPI network of common targets for the induction of testicular organoids. The circles represent proteins, the colors (from yellow to orange to red) indicate the degree of binding between the proteins. The lines represent protein-protein interactions. (H) The KEGG enrichment analyses of 109 common targets (the top ten results). (I) The Venn diagram analysis of 656 testis-specific genes and 283 predicted targets of reprogramming compounds. (J) Reprogramming compound-core gene-testis network diagram. Gray rectangles represent compounds, and circles represent genes. Red lines represent interactions between compounds and targets, and black lines represent interactions between genes.

Article Snippet: Subsequently, in the next 3 days, 200 ng/mL FGF9 (Cat# HY-P73053, MedChemExpress), 10 ng/mL BMP4 (Cat# HY-P7007, MedChemExpress), and 1 μg/mL heparin (Cat# S1346, Selleckchem) were added to the differentiation medium.

Techniques: Microarray, Expressing, Binding Assay, Protein-Protein interactions

Journal: Nature protocols

Article Title: Generation of nephron progenitor cells and kidney organoids from human pluripotent stem cells

doi: 10.1038/nprot.2016.170

Figure Lengend Snippet: Troubleshooting.

Article Snippet: We obtained the permission to use hPSCs from our Institutional Review Board (IRB) and institutional Embryonic Stem Cell Research Oversight (ESCRO) committee. human FGF2 (Peprotech, 100-18B) human FGF9 (R&D systems, 273-F9-025/CF) human Noggin (Peprotech, 120-10C) L-GlutaMAX (Life Technologies, 35050-061) O.C.T compound (Fisher Scientific, 23-730-571) Paraformaldehyde 16% (PFA, Electron Microscopy Sciences, RT15710) !

Techniques: Concentration Assay, Microscopy

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: Missense mutation in the Fgf9 gene of Eks mice. ( a ) Nucleotide sequence of the Fgf9 cDNA derived from +/+ and Eks/Eks mice. Ek s mutants have an A to C substitution at position 428, which results in the replacement of Asn143 with Thr. The Eks missense mutation is indicated by the asterisk and the corresponding amino acid is shown in purple. ( b ) Structure-based sequence alignment of human FGFs. The amino acid sequence surrounding the N143T mutation in FGF9 Eks and that of its corresponding domain in other human FGF family proteins are aligned based on sequence identity. The Asn143 residue in FGF9 is highly conserved among most FGF proteins (purple box). The asterisk denotes the site of Eks mutation.

Article Snippet: Sections through the equator of the bead were analyzed for exogenous FGF9 using goat anti-human FGF9 antibody (R&D Systems) and a cell and tissue staining kit HRP-AEC system (R&D Systems).

Techniques: Mutagenesis, Sequencing, Derivative Assay, Residue

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: Fgf9 Eks/Eks mice phenocopy Fgfr s gain-of-function mutants. ( a–l ) Defects in early specification of prospective elbow joints in Fgf9 Eks/Eks embryos. Hematoxylin and eosin staining ( a, b, g, h ) and in situ detection of Gdf5 ( c, d, i, j ) and Col2a1 ( e, f, k, l ) in the forelimb buds of Fgf9 +/+ and Fgf9 Eks/Eks embryos at E10.5 and E11.5. In Fgf9 +/+ embryos there was Gdf5 expression at the prospective elbow joint ( i ), which was demarcated as the gap of Col2a1 expression ( k , arrow), at E11.5. In Fgf9 Eks/Eks embryos there was not Gdf5 expression at the prospective elbow joint ( j ). Scale bars, 100 µm. ( m–b’ ) Ectopic osteogenesis at the coronal sutures in Fgf9 Eks/Eks fetuses. Hematoxylin and eosin staining ( m, n, u, v ) and von Kossa staining ( o, p, w, x ) and in situ detection of Spp1 ( q, r, y, z ) and Runx2 ( s, t, a’, b’ ) in the coronal suture of Fgf9 +/+ and Fgf9 Eks/Eks fetuses at E15.5 and E16.5. Note the ectopic ossification in the suture of Fgf9 Eks/Eks at E16.5 ( v, x, z, b’ ). fb, frontal bone; pb, parietal bone. Scale bars, 100 µm.

Article Snippet: Sections through the equator of the bead were analyzed for exogenous FGF9 using goat anti-human FGF9 antibody (R&D Systems) and a cell and tissue staining kit HRP-AEC system (R&D Systems).

Techniques: Staining, In Situ, Expressing

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: The Eks mutation affects dimerization of FGF9. ( a, b ) Sedimentation equilibrium analysis of FGF9 WT and FGF9 Eks . Ln A 280nm versus radius 2 during sedimentation equilibrium at 16,000 rpm at 20°C is indicated using 0.4 mg/ml of FGF9 WT ( a ) and FGF9 Eks ( b ). The residuals are shown in the upper panels. ( c, d ) Sedimentation velocity analysis of FGF9 WT and FGF9 Eks . The sedimentation coefficient distribution for FGF9 WT ( c ) and FGF9 Eks ( d ) at the concentrations of 0.2, 0.3 and 0.4 mg/ml are shown. ( e ) Analytical gel filtration chromatography of FGF9 WT and FGF9 Eks . FGF9 WT or FGF9 Eks applied separately to Superdex 75 10/300 GL columns. Eluted FGF9 WT and FGF9 Eks were identified by absorbance at 280 nm. Arrows indicate the position of the size standards: 67 kDa, albumin; 43 kDa, ovalbumin; 25 kDa, chymotrypsinogen; 13.7 kDa, ribonuclease A.

Article Snippet: Sections through the equator of the bead were analyzed for exogenous FGF9 using goat anti-human FGF9 antibody (R&D Systems) and a cell and tissue staining kit HRP-AEC system (R&D Systems).

Techniques: Mutagenesis, Sedimentation, Filtration, Chromatography

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: The Eks mutation affects the mitogenic activity of FGF9. ( a–g ) Dose dependent changes in mitogenic activity of FGF9 WT and FGF9 Eks . BaF3 cells expressing exogenous FGFR1b, 1c, 2b, 2c, 3b, 3c or 4 were treated with increasing concentrations of FGF9 WT or FGF9 Eks in the presence of 1 µg/ml heparin. Cell proliferation was determined by [ 3 H]thymidine incorporation after 36 hours in culture. ( h–n ) Heparin-dependent changes in mitogenic activity of FGF9 WT and FGF9 Eks . BaF3 cells expressing the respective FGFR were treated with increasing concentrations of heparin in the presence of 0.2 nM FGF9 WT or FGF9 Eks . Cell proliferation was determined as above. Data are represented as mean ± s.e.m. of triplicate assays. These results are representative of at least two independent experiments.

Article Snippet: Sections through the equator of the bead were analyzed for exogenous FGF9 using goat anti-human FGF9 antibody (R&D Systems) and a cell and tissue staining kit HRP-AEC system (R&D Systems).

Techniques: Mutagenesis, Activity Assay, Expressing

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: The Eks mutation reduces FGF9 affinity for heparin by impairing its dimerization. ( a ) Chromatographic analysis of the affinities of FGF9 WT and FGF9 Eks for heparin. FGF9 WT or FGF9 Eks were loaded onto a heparin-conjugated agarose column and eluted with a linear gradient of NaCl from 120 mM to 2.0 M (black line). Elution profiles of FGF9 WT and FGF9 Eks were determined by monitoring absorbance at 280 nm. ( b, c ) Surface plasmon resonance analysis of the affinities of FGF9 WT and FGF9 Eks for heparin. Sensorgrams indicating the interaction of FGF9 WT ( b ) and FGF9 Eks ( c ) with immobilized heparin were determined at different concentrations. The biosensor chip response is indicated on the y-axis (RU) as a function of time (x-axis) at room temperature. ( d–g ) The most probable solution structures of dimeric FGF9 WT -heparin ( d ), dimeric FGF9 Eks -heparin ( e ), monomeric FGF9 WT -heparin ( f ) and monomeric FGF9 Eks -heparin ( g ) complexes deduced by MD simulations. Heparin and protein residues that form important hydrogen bonds are drawn in ball and stick and space-filling modes. The single-letter amino acid code, residue number and chain code are indicated for each of these residues. Computed binding free energy of each complex is shown under the respective illustrated structure. Data are represented as mean ± s.d. of energies obtained from 200 MD snapshots in respective MD trajectory.

Article Snippet: Sections through the equator of the bead were analyzed for exogenous FGF9 using goat anti-human FGF9 antibody (R&D Systems) and a cell and tissue staining kit HRP-AEC system (R&D Systems).

Techniques: Mutagenesis, SPR Assay, Residue, Binding Assay

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: FGF9 Eks can inhibit joint and suture development as well as FGF9 WT . ( a–g ) Inhibition of knee joint development induced by ectopic expression of Fgf9 Eks as well as Fgf9 WT . Hindlimb buds of HH stage 17 chickens were infected with RCAS- Fgf9 WT , RCAS- Fgf9 Eks , or empty RCAS virus. ( a, b ) Fgf9 expression was examined by in situ hybridization 2 days after infection. ( c–g ) Respective knee joints (arrows) were examined by Alcian blue staining ( c, d, f ) and hematoxylin and eosin staining of sections through the knee joint ( e, g ) 5 days after infection. f, femur; t, tibia. ( h–k ) Inhibition of coronal suture development by the ectopic presence of FGF9 Eks well as FGF9 WT . FGF9 WT or FGF9 Eks beads were implanted onto the coronal suture at E15 mice and the Spp1 expression was examined by in situ hybridization 24 hours after implantation. On the operated sides with FGF9 WT ( h ) and FGF9 Eks ( j ) bead implants, there was overlap of Spp1 expression in the frontal and parietal bones, which was not seen on the unoperated sides ( i, k ). fb, frontal bone; pb, parietal bone.

Article Snippet: Sections through the equator of the bead were analyzed for exogenous FGF9 using goat anti-human FGF9 antibody (R&D Systems) and a cell and tissue staining kit HRP-AEC system (R&D Systems).

Techniques: Inhibition, Expressing, Infection, Virus, In Situ Hybridization, Staining

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: Ectopic FGF9 Eks signaling due to its hyper diffusibility. ( a–e ) Increased diffusibility of FGF9 Eks in the skull bone anlagen. FGF9 WT or FGF9 Eks beads were implanted onto the coronal suture at E15 mice and Spp1 expression was examined by whole-mount in situ hybridization 24 hours after implantation. On the operated sides with FGF9 WT ( a ) and FGF9 Eks ( c ) bead implants, we observed well-defined intense signals in the frontal and parietal bone anlagen around the implants, which were not seen on the unoperated side ( b, d ). This domain with intense Spp1 signals reflects diffusibility of exogenous FGF9 proteins. We therefore compared diffusibility of FGF9 WT and FGF9 Eks based on the induced expression domain of Spp1 ( e ). The diffusion areas (%) in the frontal and parietal bone anlagen area were estimated from the area ratio of the intense Spp1 expression against the frontal and parietal bone anlagen. Data are represented as mean ± s.e.m. of six operations. FGF9 Eks is more diffusible than FGF9 WT . Significance was determined by two-tailed Student’s t -test. ( f–h ) Increased diffusibility of FGF9 Eks in the forelimb bud. FGF9 WT or FGF9 Eks beads were implanted into forelimb buds of Fgf9 −/− embryos of E10.5 mice. Diffusion of exogenous FGF9 WT ( f ) and FGF9 Eks ( g ) after 2 hours was immunodetected using a FGF9 antibody. ( h ) The diffusion area of FGF9 Eks and FGF9 WT was measured at the level of the equator of the beads. Data are represented as mean (FGF9 WT =100%) ± s.e.m. of four (FGF9 WT ) or five (FGF9 Eks ) operations. FGF9 Eks is also more diffusible than FGF9 WT in limb buds. Significance was determined by one-tailed Student’s t -test. ( i–x ) The downstream target genes of FGF signaling, Etv4 and Etv5 , are expressed ectopically in the prospective elbow joint in Fgf9 Eks/Eks mice. Counterstaining with Giemsa ( i, j, q, r ) and in situ detection of Col2a1 ( k, l, s, t ), Etv4 ( m, n, u, v ) and Etv5 ( o, p, w, x ) in the forelimb buds of Fgf9 +/+ and Fgf9 Eks/Eks embryos at E10.5 and E11.5. There was ectopic expression of Etv4 ( n, v ) and Etv5 ( p, x ), in the cartilaginous condensation including the prospective elbow joint position, which was demarcated as the gap of Col2a1 expression ( s , arrow), in Fgf9 Eks/Eks , which were not seen in Fgf9 +/+ mice ( m, o, u, w ). Scale bars, 100 µm. ( y ) A model for the pathogenic mechanism underlying elbow joint synosotsis in Fgf9 Eks/Eks mice. In Fgf9 Eks/Eks mice, ectopic FGF9 signaling due to hyper-diffusion of FGF9 Eks at the prospective elbow joint may inhibit the initiation of joint development. ( z ) A model for the pathogenic mechanism underlying premature fusion of the coronal suture in Fgf9 Eks/Eks mice. In Fgf9 Eks/Eks mice, ectopic FGF9 signaling due to hyper-diffusion of FGF9 Eks at the coronal suture may promote ectopic osteogenesis and subsequently induce premature fusion of the suture.

Article Snippet: Sections through the equator of the bead were analyzed for exogenous FGF9 using goat anti-human FGF9 antibody (R&D Systems) and a cell and tissue staining kit HRP-AEC system (R&D Systems).

Techniques: Expressing, In Situ Hybridization, Diffusion-based Assay, Two Tailed Test, One-tailed Test, In Situ

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: FGF9 WT modulates FGF9 Eks action by forming FGF9 WT /FGF9 Eks heterodimers. ( a–c ) Proposed solution structures for FGF9 WT homodimer ( a ), FGF9 WT/ Eks heterodimer ( b ) and FGF9 Eks homodimer ( c ). Amino acid residues contributing to hydrogen bond formation involved in dimerization are drawn in ball and stick and space-filling modes. The single-letter amino acid code, residue number and chain code are indicated for each of these residues. Computed binding free energy of each dimer is shown under the respective illustrated structure. Data are represented as mean ± s.d. of energies obtained from 200 MD snapshots in respective MD trajectory. ( d ) FGF9 WT is capable of forming dimers with FGF9 Eks . The expression of FGF9 WT homodimers, FGF9 WT/ Eks heterodimers and FGF9 Eks homodimers was analyzed using IP/Western blots. The expression vectors for FLAG- or Myc-tagged FGF9 WT and FGF9 Eks were transfected into COS7 cells and culture supernatants were subjected to IP/Western analysis. ( e–h ) Less severe elbow joint synostosis in Fgf9 Eks/+ than Fgf9 Eks/− . Forelimbs from Fgf9 +/− , Fgf9 Eks/+ , Fgf9 Eks/− and Fgf9 Eks/Eks embryos at E17.5 were stained with Alcian blue and Alizarin red. Synostotic change is restricted to the cartilaginous component in Fgf9 Eks/+ embryos, whereas it is extended to the bony component in Fgf9 Eks/− , and Fgf9 Eks/Eks embryos. h, humerus; r, radius; u, ulna.

Article Snippet: Sections through the equator of the bead were analyzed for exogenous FGF9 using goat anti-human FGF9 antibody (R&D Systems) and a cell and tissue staining kit HRP-AEC system (R&D Systems).

Techniques: Residue, Binding Assay, Expressing, Western Blot, Transfection, Staining

Journal: Scientific reports

Article Title: Upregulation of cancer-associated gene expression in activated fibroblasts in a mouse model of non-alcoholic steatohepatitis.

doi: 10.1038/s41598-019-56039-0

Figure Lengend Snippet: Figure 3. Metabolic stress induces FGF9 expression in NASH-fib. (a) Hepatic mRNA expression levels of Fgf9 in wild-type mice fed standard diet (normal liver), Col1a2-GFP Tg mice received intraperitoneal CCl4 injection (CCl4 liver), and MC4R-KO mice fed WD for 20 weeks (NASH liver) evaluated by quantitative real-time PCR. n = 6. (b) Protein expression levels of FGF9 in normal and NASH livers by Western blot analysis. Blots are shown as cropped images. Uncropped Western blot images are included in Supplementary Fig. S2a. n = 3. *P < 0.05, **P < 0.01 vs. normal liver; †P < 0.05 vs. CCl4 liver. (c) Fgf9 mRNA expression levels in isolated HSCs and activated fibroblasts (CCl4-Fib, NASH-fib). n = 3. (d) Fgf9 mRNA expression levels in various cell types separated from normal and NASH livers. Resident macrophages, CD45+ Ly6G− F4/80hi CD11blo; recruited macrophages, CD45+ Ly6G− F4/80lo CD11bhi; CD4+ T cells, CD45+ CD4+; and liver sinusoidal endothelial cells (LSEC), CD45− CD146+. Hepatocytes were isolated from lean wild-type mice and MC4R-KO mice fed WD for 4 weeks. n = 3–8. *P < 0.05 vs. HSCs; †P < 0.05 vs. CCl4-fib. (e) mRNA expression levels in cultured HSCs treated with TGFβ (10 ng/ml), lipopolysaccharide (LPS, 10 ng/ml), and palmitic acid (200 μM) for 24 hours. (f) Dose-dependent effect of palmitate (100, 200, and 500 μM) on FGF9 induction in HSCs. (g) Effect of various fatty acids (200 μM) on FGF9 induction. Lau, laurate; Ole, oleate. n = 5. *P < 0.05, **P < 0.01 vs. veh; ##P < 0.01. Data represent mean ± SEM.

Article Snippet: The entire coding sequence of human FGF9 was amplified by FGF9 Human Tagged ORF Clone (RC210242, Origene, Rockville, MD, USA) using the primer pair set below; 5′-CACGCTACCGGTCTCGAGGCCGCCGCGATCGCCA-3′ (forward) and 5′-GCTCGACCTGCAG GATCCCTAACTTTGGCTTAGAATA-3′ (reverse).

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

Journal: Scientific reports

Article Title: Upregulation of cancer-associated gene expression in activated fibroblasts in a mouse model of non-alcoholic steatohepatitis.

doi: 10.1038/s41598-019-56039-0

Figure Lengend Snippet: Figure 4. FGF9 induces inflammatory changes in LX2 cells. Human HSC cell line LX2 cells were treated with human recombinant FGF9 at a dose of 1 or 10 ng/ml for 24 hours. (a) mRNA expression levels of proinflammatory cytokines (IL1A, IL1B), chemokines (CCL2, CXCL8) and fibrogenic factors (COL1A1, TGFB). n = 4. *P < 0.05 vs. veh. FGF9 or GFP (control)-overexpressing LX2 cells (FGF9-LX2 and control-LX2, respectively) were established using lentiviral vectors. Western blot analysis (b) and FGF9 secretion (c) into culture supernatants using FGF9-overexpressing and control-LX2 cells. Blots are shown as cropped images. Uncropped Western blot images are included in Supplementary Fig. S2b. n = 4. **P < 0.01 vs. control-LX2. (d) mRNA expression levels of genes related to proinflammatory cytokines, chemokines and fibrogenic factors in FGF9-LX2 cells. n = 8. **P < 0.01 vs. control-LX2. Data represent mean ± SEM.

Article Snippet: The entire coding sequence of human FGF9 was amplified by FGF9 Human Tagged ORF Clone (RC210242, Origene, Rockville, MD, USA) using the primer pair set below; 5′-CACGCTACCGGTCTCGAGGCCGCCGCGATCGCCA-3′ (forward) and 5′-GCTCGACCTGCAG GATCCCTAACTTTGGCTTAGAATA-3′ (reverse).

Techniques: Recombinant, Expressing, Control, Western Blot

Journal: Scientific reports

Article Title: Upregulation of cancer-associated gene expression in activated fibroblasts in a mouse model of non-alcoholic steatohepatitis.

doi: 10.1038/s41598-019-56039-0

Figure Lengend Snippet: Figure 5. FGF9 enhances cell migration and inhibits apoptosis in LX2 cells. (a) Effect of FGF9 on LX2 cell proliferation determined by WST assay after 96-hour incubation. n = 8. Effect of serum starvation (starve) for 48 hours (b) and coexistence with recombinant FGF9 (c) evaluated by caspase-3/7 activity assay in LX2 cells. n = 6. (d) Migration activity of FGF9-treated LX2 cells determined by transwell migration assay. LX2 cells were seeded onto the insert of transwell in serum free medium containing recombinant FGF9 (1 or 10 ng/ml), and incubated with medium containing 2% FBS in the lower chamber for 24 hours. n = 4. **P < 0.01 vs. starve (−) or veh. n.s., not significant. Data represent mean ± SEM.

Article Snippet: The entire coding sequence of human FGF9 was amplified by FGF9 Human Tagged ORF Clone (RC210242, Origene, Rockville, MD, USA) using the primer pair set below; 5′-CACGCTACCGGTCTCGAGGCCGCCGCGATCGCCA-3′ (forward) and 5′-GCTCGACCTGCAG GATCCCTAACTTTGGCTTAGAATA-3′ (reverse).

Techniques: Migration, WST Assay, Incubation, Recombinant, Activity Assay, Transwell Migration Assay

Journal: Scientific reports

Article Title: Upregulation of cancer-associated gene expression in activated fibroblasts in a mouse model of non-alcoholic steatohepatitis.

doi: 10.1038/s41598-019-56039-0

Figure Lengend Snippet: Figure 6. FGF9 enhances cell migration and inhibits apoptosis in HepG2 cells. (a) Effect of FGF9 on HepG2 cell proliferation determined by WST assay after 96-hour incubation. n = 8. Effect of anti-Fas antibody (Fas) at a dose of 100 ng/ml for 24 hours (b) and coexistence with recombinant FGF9 (c) evaluated by caspase-3/7 activity assay in HepG2 cells. n = 6. (d) Migration activity of FGF9-treated HepG2 cells determined by transwell migration assay. HepG2 cells were seeded onto the insert of transwell in serum free medium containing recombinant FGF9 (1 or 10 ng/ml), and incubated with medium containing 2% FBS in the lower chamber for 24 hours. n = 4. **P < 0.01 vs. veh. n.s., not significant. Data represent mean ± SEM.

Article Snippet: The entire coding sequence of human FGF9 was amplified by FGF9 Human Tagged ORF Clone (RC210242, Origene, Rockville, MD, USA) using the primer pair set below; 5′-CACGCTACCGGTCTCGAGGCCGCCGCGATCGCCA-3′ (forward) and 5′-GCTCGACCTGCAG GATCCCTAACTTTGGCTTAGAATA-3′ (reverse).

Techniques: Migration, WST Assay, Incubation, Recombinant, Activity Assay, Transwell Migration Assay

Journal: Scientific reports

Article Title: Upregulation of cancer-associated gene expression in activated fibroblasts in a mouse model of non-alcoholic steatohepatitis.

doi: 10.1038/s41598-019-56039-0

Figure Lengend Snippet: Figure 7. FGF9 promotes tumor growth in a human tumor xenograft model. HepG2 cells (2 × 105 cells) together with control-LX2 or FGF9-LX2 cells (1 × 106 cells) were transplanted subcutaneously in the flank of immunodeficient mice. (a) Time course of the tumor volume. Weight (b) and representative images (c) of subcutaneous tumors at 4 weeks after transplantation. αSMA staining (d) and TUNEL staining (e) of the tumors. Arrows indicate TUNEL-positive cells. (f) GFP and TUNEL double immunofluorescent staining of the tumors that includes HepG2 cells and control (GFP)-LX2 cells. (g) Ki67 immunostaining of the tumors. Scale bars: 100 μm. *P < 0.05, **P < 0.01 vs. control. n = 7. Data represent mean ± SEM.

Article Snippet: The entire coding sequence of human FGF9 was amplified by FGF9 Human Tagged ORF Clone (RC210242, Origene, Rockville, MD, USA) using the primer pair set below; 5′-CACGCTACCGGTCTCGAGGCCGCCGCGATCGCCA-3′ (forward) and 5′-GCTCGACCTGCAG GATCCCTAACTTTGGCTTAGAATA-3′ (reverse).

Techniques: Control, Transplantation Assay, Staining, TUNEL Assay, Immunostaining