Journal: eLife

Article Title: Hair follicle dermal condensation forms via Fgf20 primed cell cycle exit, cell motility, and aggregation

doi: 10.7554/eLife.36468

Figure Lengend Snippet: ( A ) Quantification of scratch wound closure of E13.5 growth-arrested primary dermal fibroblasts treated with FGF9 (200 ng/ml) or FGF9 and SU5402 (20 µM). At 24 hr, FGF9 treatment resulted in greater wound-closure at 8 hr (36.17 ± 5.04%, p=0.0490) and at 24 hr (97.11 ± 3.09%, p=0.0133) compared to DMSO control (all treatments n = 5 experiments, each performed with freshly extracted primary dermal fibroblast cell population). Wound closure was not altered when cells were treated with both FGF9 and SU5402 inhibitor (p=0.2563). ( B ) Quantification of transwell migration assay of E13.5 primary dermal fibroblasts. Migration was significantly increased when FGF9 (200 ng/ml) was added to lower or both upper and lower chambers (p=0.0003 and p=0.0010, respectively; for both n = 6 experiments, each performed with freshly extracted primary dermal fibroblast cell population). No statistical difference was observed between FGF9 treatments (p=0.4033). ( C ) Quantification of nuclei density in E13.5 dermis explants treated for 3 hr with beads loaded with FGF9 (100 µg/ml) or 0.1% BSA vehicle control. Density measured from a single optical slice at mid-bead. FGF9 bead induces an increase in density within 15 µm radius from the bead relative to BSA control (p=0.033, n = 7 explants), but not between 15 and 30 µm radius (p=0.236, n = 7 explants). ( D ) Whole-mount RNA in situ hybridization of dermal samples treated with FGF9 beads for 3, 8, and 16 hr. Induction of Spry4 and Dusp6 , but not Sox2 expression (purple) was observed around the bead at all time points ( n indicates induction/total samples, induction was tested in two independent experiments with skin samples derived from at least two different litters.). ( E ) 2 hr EdU incorporation into dermis organ cultures after overnight incubation with BSA (left), FGF20 (center), FGF9 (right) loaded beads. Note the increased number of proliferating cells around the FGF9 bead ( n = 5 explants). Error bars represent SD. *, p≤0.05; **, p≤0.01; ***, p≤0.001. Scale bar = 30 µm. See also and . 10.7554/eLife.36468.027 Figure 6—figure supplement 1—source data 1. Values used to quantify FGF9-induced cellular changes. Values used to quantify fibroblast wound closure in the presence of DMSO, SU5402, FGF9 +DMSO, or FGF9 +SU5402 . Values used to quantify E13.5 primary fibroblast transwell migration in control, FGF9 in lower chamber, and FGF9 in seeding and lower chambers . Values used to quantify fibroblast density in response to BSA or FGF9-loaded beads at 0–15 µm and 15–30 µm distance from the bead .

Article Snippet: Peptide, recombinant protein , FGF9 human recombinant protein , R and D Systems , 273-F9 , .

Techniques: Control, Transwell Migration Assay, Migration, RNA In Situ Hybridization, Expressing, Derivative Assay, Incubation

Journal: eLife

Article Title: Hair follicle dermal condensation forms via Fgf20 primed cell cycle exit, cell motility, and aggregation

doi: 10.7554/eLife.36468

Figure Lengend Snippet: 3 hr incubation of BSA-, FGF20- or FGF9-loaded beads with E13.5 wildtype ( A, B, C ) or Fucci mKO ( D ) dermises. Cdkn1a expression was assayed in these samples using ( A ) whole-mount RNA in situ hybridization of dermal samples. Cdkn1a expression (purple) was not observed around the bead (0/20 each condition, in four independent experiments with skin samples derived from four different litters.) ( B ) section radioactive in situ hybridization (0/5 each condition in two independent experiments with skin samples from two different litters), and ( C ) section in situ hybridization (0/5 each condition in two independent experiments with skin samples from two different litters). ( D ) Cell cycle exit was assessed using Fucci mKO reporter allele (representing G 0 /G 1 cell cycle phase) in the 30 µm surrounding the center of the bead (0/10 each condition in two independent experiments with skin samples from two different litters). ( E ) Quantification of percent total cells surrounding the bead positive for Fucci-mKO . No significant difference was observed between any of the groups. Error bars represent SD. Scale bar = 50 µm. See also and . 10.7554/eLife.36468.029 Figure 6—figure supplement 2—source data 1. Values used to quantify FGF20 or FGF9 induced Fucci-mKO expression. Values used to quantify the percent of total cells expressing Fucci-mKO 30 µm surrounding the center of the bead .

Article Snippet: Peptide, recombinant protein , FGF9 human recombinant protein , R and D Systems , 273-F9 , .

Techniques: Incubation, Expressing, RNA In Situ Hybridization, Derivative Assay, In Situ Hybridization

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: 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 CCl 4 injection (CCl 4 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. . n = 3. * P < 0.05, ** P < 0.01 vs. normal liver; † P < 0.05 vs. CCl 4 liver. ( c ) Fgf9 mRNA expression levels in isolated HSCs and activated fibroblasts (CCl 4 -Fib, NASH-fib). n = 3. ( d ) Fgf9 mRNA expression levels in various cell types separated from normal and NASH livers. Resident macrophages, CD45 + Ly6G − F4/80 hi CD11b lo ; recruited macrophages, CD45 + Ly6G − F4/80 lo CD11b hi ; 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. CCl 4 -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′-GCTCGACCTGCAGGATCCCTAACTTTGGCTTAGAATA-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: 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. . 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′-GCTCGACCTGCAGGATCCCTAACTTTGGCTTAGAATA-3′ (reverse).

Techniques: Recombinant, Expressing, 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: 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′-GCTCGACCTGCAGGATCCCTAACTTTGGCTTAGAATA-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: 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′-GCTCGACCTGCAGGATCCCTAACTTTGGCTTAGAATA-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: FGF9 promotes tumor growth in a human tumor xenograft model. HepG2 cells (2 × 10 5 cells) together with control-LX2 or FGF9-LX2 cells (1 × 10 6 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′-GCTCGACCTGCAGGATCCCTAACTTTGGCTTAGAATA-3′ (reverse).

Techniques: Transplantation Assay, Staining, TUNEL Assay, Immunostaining