Journal: International Journal of Biological Sciences

Article Title: Tubulointerstitial nephritis antigen-like 1 promotes the progression of liver fibrosis after HCV eradication with direct-acting antivirals

doi: 10.7150/ijbs.103305

Figure Lengend Snippet: TINAGL1 is increased in HCV-infected patients, hepatocytes and in the liver of mice and patients with fibrosis. (A) Venn diagram analysis of genes in HCV-infected Huh7.5 cells at the indicated time points (Fold change ≥ 2). (B-C) RNA levels quantified by qRT-PCR (B) and proteins detected by Western blot (C) in Huh7.5 cells infected with HCV for over three months ( n = 3). (D-E) RNA (D) and protein (E) levels in Huh7.5 cells infected with HCV (MOI = 0.1) for 24, 48, and 72 hours ( n = 3). (F-G) RNA (F) and protein (G) levels in Huh7.5 cells infected with HCV (MOI = 0.01, 0.1, and 1) for 72 hours ( n = 3). (H) TINAGL1 quantified by ELISA in the culture medium of HCV-infected Huh7.5 cells ( n = 3). (I) TINAGL1 mRNA in liver biopsies from Gene Expression Omnibus database (GSE38226). (J) TINAGL1 measured by Western blot in mouse livers with fibrosis. (K-L) Statistical summary of TINAGL1 expression in a human liver tissue array from patients with MASH ( n = 29), fibrosis ( n = 92), cirrhosis ( n = 30), and normal donors ( n = 10). Data were expressed as mean ± standard deviation. P values were calculated by an unpaired two-tailed Student's t-test (B, H, I, and L) or one-way ANOVA (D, F, and K) using Tukey's multiple comparisons test. α-SMA, alpha-smooth muscle actin; CCl 4 , carbon tetrachloride; DEN, diethylnitrosamine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HCV, hepatitis C virus; TINAGL1, tubulointerstitial nephritis antigen-like 1.

Article Snippet: The human TINAGL1 ELISA kit (Boster, EK1766) was used to examine TINAGL1 concentration in culture medium and in cell lysates of Huh7.5 cells.

Techniques: Infection, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Gene Expression, Expressing, Standard Deviation, Two Tailed Test, Virus

Journal: International Journal of Biological Sciences

Article Title: Tubulointerstitial nephritis antigen-like 1 promotes the progression of liver fibrosis after HCV eradication with direct-acting antivirals

doi: 10.7150/ijbs.103305

Figure Lengend Snippet: TINAGL1 expression remains higher in hepatocytes after HCV elimination by high-efficiency treatment with DAAs . (A) Schematic diagram (left) for the establishment of HCV-eradicated Huh7.5 cells and HCV RNA (right) in the cells ( n = 3). (B) TINAGL1 quantified by ELISA in the culture medium of HCV-eradicated Huh7.5 cells ( n = 3). (C-D) mRNA (C) and protein (D) of TINAGL1 in HCV-eradicated Huh7.5 cells ( n = 3). (E) Protein levels in HCV-eradicated Huh7.5 cells after re-infection with HCV for 72 hours ( n = 3). Data were expressed as mean ± standard deviation. P values were calculated by one-way ANOVA (B, C) using Tukey's multiple comparisons test. DAAs, direct-acting antivirals.

Article Snippet: The human TINAGL1 ELISA kit (Boster, EK1766) was used to examine TINAGL1 concentration in culture medium and in cell lysates of Huh7.5 cells.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Infection, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: Tubulointerstitial nephritis antigen-like 1 promotes the progression of liver fibrosis after HCV eradication with direct-acting antivirals

doi: 10.7150/ijbs.103305

Figure Lengend Snippet: TINAGL1 promotes the activation of hepatic stellate cells in vitro. (A) Schematic diagram of the transwell insert mono- and co-culture models. (B-C) mRNA levels in LX-2 cells co-cultured with HCV-infected Huh7.5 cells (B) or their CM (C) ( n = 5). (D-E) mRNA levels in LX-2 cells co-cultured with Huh7.5 cells transfected with the TINAGL1 plasmid (D) or their CM (E) ( n = 5). (F) mRNA levels in LX-2 cells treated with rhTINAGL1 for 48 hours ( n = 3). Data were expressed as mean ± standard deviation. P values were calculated by an unpaired two-tailed Student's t-test (B-E) or one-way ANOVA (F) using Tukey's multiple comparisons test. ACTA2 (α-SMA), alpha-smooth muscle actin; CM conditioned medium; COL1A1 , collagen type I alpha 1; rhTINAGL1, recombinant human tubulointerstitial nephritis antigen-like 1; TIMP1 , tissue inhibitor of matrix metalloproteinase 1.

Article Snippet: The human TINAGL1 ELISA kit (Boster, EK1766) was used to examine TINAGL1 concentration in culture medium and in cell lysates of Huh7.5 cells.

Techniques: Activation Assay, In Vitro, Co-Culture Assay, Cell Culture, Infection, Transfection, Plasmid Preparation, Standard Deviation, Two Tailed Test, Recombinant

Journal: International Journal of Biological Sciences

Article Title: Tubulointerstitial nephritis antigen-like 1 promotes the progression of liver fibrosis after HCV eradication with direct-acting antivirals

doi: 10.7150/ijbs.103305

Figure Lengend Snippet: TINAGL1 activates HSCs by stabilizing PDGF-BB. (A) Scatter Chart of fibrosis-associated cytokines in the culture medium of Huh7.5 cells transfected with the TINAGL1 plasmid detected by a cytokine microarray assay. (B) PDGF-BB concentration quantified by ELISA in cell lysate and culture medium and by Western Blot in cell lysate of Huh7.5 cells transfected with the TINAGL1 plasmid ( n = 3). (C) PDGFB mRNA level in Huh7.5 cells transfected with the TINAGL1 plasmid ( n = 4). (D) Protein levels in Huh7.5 cells transfected with the TINAGL1-Flag plasmid and treated with cycloheximide (CHX), the intensity of protein was scanned by Image J ( n =3). (E) Binding mode between human TINAGL1 and PDGF-BB. (F) Binding affinity between rhTINAGL1 (C-6 × His) and rhPDGF-BB proteins detected by SPR method. (G) Interaction of TINAGL1 and PDGF-BB in HEK293T cells detected by co-immunoprecipitation. (H) Co-localization of TINAGL1 (red) and PDGF-BB (green) in HEK293T cells (Scale bar: 5 μm). Nuclear (blue). Statistical analysis of co-localization of TINAGL1 and PDGF-BB fluorescence intensities was performed using ImageJ software. Data were expressed as mean ± standard deviation. P values were calculated by an unpaired two-tailed Student's t-test (B-D). ACTB, beta-actin; CHX, cycloheximide; PDGF-BB, platelet-derived growth factor BB; rhPDGF-BB, recombinant human platelet-derived growth factor BB; rhTINAGL1, recombinant human tubulointerstitial nephritis antigen-like 1; SPR, surface plasmon resonance.

Article Snippet: The human TINAGL1 ELISA kit (Boster, EK1766) was used to examine TINAGL1 concentration in culture medium and in cell lysates of Huh7.5 cells.

Techniques: Transfection, Plasmid Preparation, Microarray, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Binding Assay, Immunoprecipitation, Fluorescence, Software, Standard Deviation, Two Tailed Test, Derivative Assay, Recombinant, SPR Assay

Journal: International Journal of Biological Sciences

Article Title: Tubulointerstitial nephritis antigen-like 1 promotes the progression of liver fibrosis after HCV eradication with direct-acting antivirals

doi: 10.7150/ijbs.103305

Figure Lengend Snippet: TINAGL1 activates HSCs by PDGF-BB/PDGFRβ pathway. (A) PDGFRB mRNA levels in LX-2 cells treated with rhTINAGL1 for 48 hours ( n = 3). (B-C) Protein levels of α-SMA and PDGFRβ in LX-2 cell and TINAGL1 in cell medium after co-culture with Huh7.5 cells transfected with TINAGL1 plasmid, siRNA (B) or neutralizing antibody (C) against PDGFRβ, the intensity of protein was scanned by Image J ( n =3). Data were expressed as mean ± standard deviation. P values were calculated by one-way ANOVA (A-C) using Tukey's multiple comparisons test. α-SMA, alpha-smooth muscle actin; PDGFRβ, platelet-derived growth factor receptor beta; rhTINAGL1, recombinant human tubulointerstitial nephritis antigen-like 1.

Article Snippet: The human TINAGL1 ELISA kit (Boster, EK1766) was used to examine TINAGL1 concentration in culture medium and in cell lysates of Huh7.5 cells.

Techniques: Co-Culture Assay, Transfection, Plasmid Preparation, Standard Deviation, Derivative Assay, Recombinant

Journal: International Journal of Biological Sciences

Article Title: Tubulointerstitial nephritis antigen-like 1 promotes the progression of liver fibrosis after HCV eradication with direct-acting antivirals

doi: 10.7150/ijbs.103305

Figure Lengend Snippet: Liver-specific overexpression of TINAGL1 initiates and exacerbates liver fibrosis in mice. (A) Schematic overview of the experimental setup for (B-C) . (B) H&E staining (Scale bar: 100 μm), Masson staining (Scale bar: 100 μm), and Sirius red staining (Scale bar: 100 μm) of mouse livers. Quantification of Masson and Sirius red staining with Image J. (C) mRNA and protein levels in mouse livers ( n = 5). (D) Schematic overview of the experimental setup for (E-I). (E) H&E staining (Scale bar: 100 μm), Masson staining (Scale bar: 100 μm), and Sirius red staining (Scale bar: 100 μm) of mouse livers. (F) AST and ALT in the serum of mice ( n = 5). (G) Hydroxyproline content in mouse livers ( n = 5). (H) Liver / body weight ratio ( n = 5). (I) Protein levels in mouse livers. Data were expressed as mean ± standard deviation. P values were calculated by an unpaired two-tailed Student's t-test (B-C) or one-way ANOVA (F-H) using Tukey's multiple comparisons test. Acta2 (α-SMA), alpha-smooth muscle actin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CCl 4 , carbon tetrachloride; COL1A1, collagen type I alpha 1; PDGFRβ, platelet-derived growth factor receptor beta; TIMP1, tissue inhibitor of matrix metalloproteinase 1.

Article Snippet: The human TINAGL1 ELISA kit (Boster, EK1766) was used to examine TINAGL1 concentration in culture medium and in cell lysates of Huh7.5 cells.

Techniques: Over Expression, Staining, Standard Deviation, Two Tailed Test, Derivative Assay

Journal: International Journal of Biological Sciences

Article Title: Tubulointerstitial nephritis antigen-like 1 promotes the progression of liver fibrosis after HCV eradication with direct-acting antivirals

doi: 10.7150/ijbs.103305

Figure Lengend Snippet: Liver-specific knockdown of TINAGL1 prevents the progression of liver fibrosis in mice induced by CCl 4 . (A) Schematic overview of the experimental setup. (B) mRNA level of Tinagl1 in mouse livers ( n = 5). (C) Body weight ( n = 5). (D) H&E staining (Scale bar: 100 μm), Masson staining (Scale bar: 100 μm), and Sirius Red staining (Scale bar: 100 μm) of mouse livers. Quantification of Masson and Sirius red staining with Image J. (E) Liver / body weight ratio ( n = 5). (F) Hydroxyproline content in mouse livers ( n = 5). (G) Serum AST and ALT in mice ( n = 5). (H) mRNA and protein levels in mouse livers ( n = 5). (I) Serum PDGF-BB level quantified by ELISA ( n = 5). Data were expressed as mean ± standard deviation. P values were calculated by one-way ANOVA (B, D-I) using Tukey's multiple comparisons test or two-way ANOVA (C) using Bonferroni's multiple comparisons test. Acta2 (α-SMA), alpha-smooth muscle actin; AST, aspartate aminotransferase; ALT alanine aminotransferase; COL1A1, collagen type I alpha 1; PDGFRβ, platelet-derived growth factor receptor beta; TIMP1, tissue inhibitor of matrix metalloproteinase 1; Il1b , interleukin-1 beta; Il6, interleukin 6; Tnfa , tumor necrosis factor alpha.

Article Snippet: The human TINAGL1 ELISA kit (Boster, EK1766) was used to examine TINAGL1 concentration in culture medium and in cell lysates of Huh7.5 cells.

Techniques: Knockdown, Staining, Enzyme-linked Immunosorbent Assay, Standard Deviation, Derivative Assay

Journal: International Journal of Biological Sciences

Article Title: Tubulointerstitial nephritis antigen-like 1 promotes the progression of liver fibrosis after HCV eradication with direct-acting antivirals

doi: 10.7150/ijbs.103305

Figure Lengend Snippet: Liver-specific knockdown of TINAGL1 alleviates liver fibrosis in mice induced by CCl 4 . (A) Schematic overview of the experimental setup. (B) mRNA level of Tinagl1 in mouse livers ( n = 5). (C) Body weight ( n = 5). (D) H&E staining (Scale bar: 100 μm), Masson staining (Scale bar: 100 μm), and Sirius Red staining (Scale bar: 100 μm) of mouse livers. Quantification of Masson and Sirius red staining with Image J. (E) Liver / body weight ratio ( n = 5). (F) Hydroxyproline content in mouse livers ( n = 5). (G) Serum ALT and AST in mice ( n = 5). (H) mRNA and protein levels in mouse livers ( n = 5). (I) Serum PDGF-BB level quantified by ELISA ( n = 5). Data were expressed as mean ± standard deviation. P values were calculated by one-way ANOVA (B, D-I) using Tukey's multiple comparisons test or two-way ANOVA (C) using Bonferroni's multiple comparisons test. Acta2 (α-SMA), alpha-smooth muscle actin; AST, aspartate aminotransferase; ALT alanine aminotransferase; COL1A1, collagen type I alpha 1; PDGFRβ, platelet-derived growth factor receptor beta; TIMP1, tissue inhibitor of matrix metalloproteinase 1; Il1b , interleukin-1 beta; Il6, interleukin 6; Tnfa , tumor necrosis factor alpha.

Article Snippet: The human TINAGL1 ELISA kit (Boster, EK1766) was used to examine TINAGL1 concentration in culture medium and in cell lysates of Huh7.5 cells.

Techniques: Knockdown, Staining, Enzyme-linked Immunosorbent Assay, Standard Deviation, Derivative Assay