Journal: bioRxiv

Article Title: Thrombospondin 2 is a key determinant of fibrogenesis in NAFLD

doi: 10.1101/2023.06.01.543250

Figure Lengend Snippet: a-c, Gene expression analysis using a NAFLD liver-derived microarray dataset (GSE49541). Number of genes (a, left) , volcano plot (a, right) , and mean differentiation plot (b) with an adjusted P value of <0.05 when comparing F3-4 cases with F0-1 cases. The large red dot indicates THBS2. (c) THBS2 expression levels by NAFLD case in GSE49541 (F0-1, n=40; F3-4, n=32). d, e, Relative THBS2 mRNA expression levels in NAFLD liver tissue samples from our institution. Relative THBS2 mRNA expression levels (n=96) by fibrosis stage (d) , F0-1 (n=59) vs. F2-4 (n=37) and F0-2 (n=71) vs. F3-4 (n=25) (e) . Data are presented as the mean ± standard error of the mean. Correlation analysis was conducted by Spearman’s test (d) or the two-tailed Student’s t -test (e) .

Article Snippet: Human THBS2 siRNA and control siRNA were purchased from Santa Cruz.

Techniques: Gene Expression, Derivative Assay, Microarray, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Thrombospondin 2 is a key determinant of fibrogenesis in NAFLD

doi: 10.1101/2023.06.01.543250

Figure Lengend Snippet: a, b , Enrichment analysis (a) and network analysis (b) in a NAFLD liver gene set (GSE49541) using Metascape . (b) Group of genes showing strong linkage to THBS2. This is a partial expansion of . c, d, Correlation of relative mRNA expression levels of THBS2 and COL1A1 (c) , as well as of THBS2 with COL1A2, COL3A1, and COL4A1 (d) in NAFLD liver tissue samples from our institution (n=96). Correlation analysis was conducted by Spearman’s test (c) .

Article Snippet: Human THBS2 siRNA and control siRNA were purchased from Santa Cruz.

Techniques: Expressing

Journal: bioRxiv

Article Title: Thrombospondin 2 is a key determinant of fibrogenesis in NAFLD

doi: 10.1101/2023.06.01.543250

Figure Lengend Snippet: a-c, GSE174748 and GSE189175 scRNA-seq datasets were used in this study. See Methods for details. (a) Liver cell clusters in the whole dataset. b, c, THBS2 expression levels in each cell cluster (b) and in HSCs (c) . THBS2 expression levels of HSCs are shown in three groups: Healthy, NAFLD with fibrosis, and NAFLD without fibrosis.

Article Snippet: Human THBS2 siRNA and control siRNA were purchased from Santa Cruz.

Techniques: Expressing

Journal: bioRxiv

Article Title: Thrombospondin 2 is a key determinant of fibrogenesis in NAFLD

doi: 10.1101/2023.06.01.543250

Figure Lengend Snippet: a-d, In situ hybridization analysis of liver tissue samples using a THBS2 mRNA probe (left) and Azan-Mallory staining (right). NAFLD cases with fibrosis (F3, n=3) ( a-c ) and control case without chronic liver disease (n=1) ( d ). Arrows indicate representative positive sites.

Article Snippet: Human THBS2 siRNA and control siRNA were purchased from Santa Cruz.

Techniques: In Situ Hybridization, Staining, Control

Journal: bioRxiv

Article Title: Thrombospondin 2 is a key determinant of fibrogenesis in NAFLD

doi: 10.1101/2023.06.01.543250

Figure Lengend Snippet: a, COL1A1 mRNA expression levels in HepG2 and LX-2 cells in the presence and absence (control) of TGFβ. b, COL1A1 mRNA expression levels at different TGFβ concentrations. c, THBS2 mRNA expression levels in HepG2 and LX-2 cells in the presence and absence (control) of TGFβ. d, Western blotting studies with LX-2 cells treated in the presence and absence (control) of TGFβ. e-g, Quantitative analysis of TSP2 protein expression levels in cell lysates by western blotting (e) and ELISA (f), as well as in the supernatant (g) by ELISA of LX-2 cells in the presence and absence (control) of TGFβ a, c, Data were expressed relative to basal levels measured in the absence of TGFβ with HepG2 cells. b, Data were expressed relative to basal levels measured in the absence of TGFβ. e-g, TSP2 protein expression levels were normalized relative to protein expression levels of the control. TGFβ treatment was 4 ng/mL for 10 hours, except for b. Data are presented as the mean ± standard error of the mean of at least three independent experiments. Correlation analysis was conducted by one-way ANOVA followed by Bonferroni’s post-hoc test (a, c) and the two-tailed Student’s t -test (e-g) .

Article Snippet: Human THBS2 siRNA and control siRNA were purchased from Santa Cruz.

Techniques: Expressing, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Thrombospondin 2 is a key determinant of fibrogenesis in NAFLD

doi: 10.1101/2023.06.01.543250

Figure Lengend Snippet: a, b, Western blotting studies (a) and relative THBS mRNA expression levels (b) in LX-2 cells treated with control siRNA and THBS2 siRNA. c, d, Relative COL1A1 mRNA expression levels treated with control siRNA and THBS2 siRNA in the presence (d) and absence (c) of TGFβ. e, Relative COL1A2 mRNA expression levels in the presence and absence of recombinant TSP2 protein (30 ng/mL). f, Fluorescent staining of LX-2 cells treated with control siRNA or THBS2 siRNA or in the presence and absence of TGFβ: (i) control siRNA, (ii) THBS2 siRNA, (iii) control siRNA and TGFβ, and (iv) THBS2 siRNA and TGFβ. Collagen expression was identified as green and cell nuclei as blue DAPI. g, Relative quantification of collagen expression values per cell. h, Expression levels of THBS2 mRNA and COL1A1 mRNA over time after addition of TGFβ to LX-2 cells. b-e, g, Data were normalized relative to mRNA expression levels of the control. TGFβ treatment was performed with 4 ng/mL for 10 hours, except for h. Data are presented as the mean ± standard error of the mean of at least three independent experiments. Correlation analysis was conducted by the two-tailed Student’s t -test (b-e) and one-way ANOVA followed by Bonferroni’s post-hoc test (g) .

Article Snippet: Human THBS2 siRNA and control siRNA were purchased from Santa Cruz.

Techniques: Western Blot, Expressing, Control, Recombinant, Staining, Quantitative Proteomics, Two Tailed Test

Journal: bioRxiv

Article Title: Thrombospondin 2 is a key determinant of fibrogenesis in NAFLD

doi: 10.1101/2023.06.01.543250

Figure Lengend Snippet: a, b, Western blotting studies of SMAD 2/3 (a) and quantitative analysis (b) in LX-2 cells treated with control siRNA and THBS2 siRNA in the presence and absence of TGFβ. Control: control siRNA, siRNA: THBS2 siRNA. c, d, SMAD inhibitors downregulate COL1A1 mRNA expression levels without affecting THBS2 mRNA expression levels. SMADi: SMAD inhibitor, SIS3 (10 µM). d, Proposed mechanism of TGFβ-THBS2/TSP2 expression in LX-2 cells. b, c, Data were normalized relative to expression levels of the control. TGFβ treatment was performed with 4 ng/mL for 10 hours. Data are presented as the mean ± standard error of the mean of at least three independent experiments. Correlation analysis was conducted by one-way ANOVA followed by Bonferroni’s post-hoc test (b) and the two-tailed Student’s t -test (c) .

Article Snippet: Human THBS2 siRNA and control siRNA were purchased from Santa Cruz.

Techniques: Western Blot, Control, Expressing, Two Tailed Test