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