Journal: Journal of hepatology

Article Title: TAZ-induced Cybb contributes to liver tumor formation in non-alcoholic steatohepatitis

doi: 10.1016/j.jhep.2021.11.031

Figure Lengend Snippet: (A) Livers (arrows, tumors) and liver sections of mice fed the NASH diet for 15 months. The sections were stained with H&E (imaged at 4X and 20X; bars, 1 mm and 200 μm, respectively; T, tumor) and with reticulin and ant-iglypican-3 (bars, 100 μm). (B-E) Mice were fed the NASH diet for 13 months, with AAV8-H1-shTaz (shTaz) or control vector (Scr) administered at 8 monts. (B) Experimental scheme. (C) TAZ immunoblot from non-tumor liver tissue. (D) Livers (arrows, tumors) and tumor numbers/mouse. (E) Liver sections from non-tumor areas stained for Ki67 (green) and HNF4α (red) and quantified for the percent Ki67 + HNF4α + cells (arrows, Ki67 + HNF4α + cells; bar, 100 μm). For D-E, n = 6 mice/group; means ± SEM; *p < 0.05 by Student’s t-test. (F) Livers (arrows, tumors) and liver sections of mice that were administered DMBA post-natal day 5; placed on NASH diet at 1 month of age, and analyzed at 9 months. The sections were stained with H&E, Sirius red (bars, 500 μm; T, tumor), reticulin, and anti-glypican-3 (bars, 200 μm). (G-J) Wwtr1 fl/fl male mice were administered DMBA on postnatal day 5; placed on the NASH diet at 1 month; injected with AAV8-TBG-LacZ or AAV8-TBG-Cre at 5 months; and analyzed at 9 months. (G) Experimental scheme. (H) TAZ immunoblot from non-tumor liver tissue. (I) Livers (arrows, tumors) and tumor numbers and average diameter. (J) Percent Ki67 + HNF4α + cells in non-tumor areas. For I-J, n = 5 mice/group; means ± SEM; *p < 0.05 by Student’s t-test. (K) Livers (arrows, tumors) and liver sections of Rosa NICD mice injected with AAV8-TBG-Cre to activate hepatocyte Notch; started on the NASH diet 1 week later; and analyed after 4 months on diet. The sections were stained with H&E, Sirius red (bars, 500 μm), reticulin, and anti-glypican-3 (bars, 200 μm). (L-O) Rosa NICD or Rosa NICD Wwtr1 fl/fl mice were treated with AAV8-TBG-Cre; placed on NASH diet 1 week later; and analyzed 4 months later. (L) Experimental scheme. (M) TAZ immunoblot from non-tumor liver tissue. (N) Livers (arrows, tumors) and tumor numbers and average diameter. (O) Percent Ki67 + HNF4α + cells in non-tumor areas. For N-O, n = 5 mice/group; means ± SEM; *p < 0.05 by Student’s t-test.

Article Snippet: Male wild-type C57BL/6J mice (#000664, 9–10 weeks/old), Cybb fl/fl mice (#031777), and Rosa NICD mice (#008159) were from Jackson Laboratory (Bar Harbor, ME) and allowed to adapt in the animal facility for 1 week prior to random assignment to experimental cohorts.

Techniques: Staining, Control, Plasmid Preparation, Western Blot, Injection

Journal: Journal of hepatology

Article Title: TAZ-induced Cybb contributes to liver tumor formation in non-alcoholic steatohepatitis

doi: 10.1016/j.jhep.2021.11.031

Figure Lengend Snippet: (A-D) AAV8-TBG-Cre-treated Rosa NICD mice were fed the NASH diet and, 3 months later, injected with AAV8-H1-scrambled RNA or AAV8-H1-shTaz. The mice were analyzed at month 5. (A) Experimental scheme. (B) TAZ immunoblot from surrounding tissue (ST) and tumor tissue. (C) Livers (arrows, tumors) and tumor numbers and average diameter. (D) Quantification of the percentage of Ki67 + HNF4α + cells in liver sections from non-tumor areas. For C-D, n = 7–8 mice/group; means ± SEM; *p < 0.05 by Student’s t-test.

Article Snippet: Male wild-type C57BL/6J mice (#000664, 9–10 weeks/old), Cybb fl/fl mice (#031777), and Rosa NICD mice (#008159) were from Jackson Laboratory (Bar Harbor, ME) and allowed to adapt in the animal facility for 1 week prior to random assignment to experimental cohorts.

Techniques: Injection, Western Blot

Journal: Journal of hepatology

Article Title: TAZ-induced Cybb contributes to liver tumor formation in non-alcoholic steatohepatitis

doi: 10.1016/j.jhep.2021.11.031

Figure Lengend Snippet: (A-F) AAV8-TBG-Cre-treated Rosa NICD mice were fed the NASH diet and, 2 months later, injected with AAV8-H1-shTaz and either AAV8-TBG-LacZ or AAV8-TBG-Ihh. The mice were analyzed at month 4. (A) Experimental scheme. (B) Ihh immunoblot from surrounding tissue (ST) and tumor tissue. (C) Liver sections stained with H&E (upper images) and Sirius red (lower images), with quantification of inflammatory cells and percent Sirius red-positive area. Bars, 200 μm. (D) Percent TUNEL + cells from non-tumor areas. (E) Plasma ALT. (F) Livers (arrows, tumors) and tumor numbers and diameter. For C-F, n = 6 mice/group; means ± SEM; *p < 0.05 by Student’s t-test. (G-L) AAV8-TBG-Cre-treated Rosa NICD mice were fed the NASH diet and, 2 months later, injected with AAV8-H1-Scr or AAV8-H1-shIhh. The mice were analyzed at month 4. (G) Experimental scheme. (H) Ihh immunoblot from surrounding tissue (ST) and tumor tissue. (I) Liver sections were stained with H&E (upper images) and Sirius red (lower images), with quantification of inflammatory cells and percent Sirius red-positive area. Bars, 200 μm. (J) Percent TUNEL + cells from non-tumor areas. (K) Plasma ALT. (L) Livers (arrows, tumors) and tumor numbers and diameter. For I-L, n = 6–7 mice/group; means ± SEM; *p < 0.05 by Student’s t-test.

Article Snippet: Male wild-type C57BL/6J mice (#000664, 9–10 weeks/old), Cybb fl/fl mice (#031777), and Rosa NICD mice (#008159) were from Jackson Laboratory (Bar Harbor, ME) and allowed to adapt in the animal facility for 1 week prior to random assignment to experimental cohorts.

Techniques: Injection, Western Blot, Staining, TUNEL Assay, Clinical Proteomics

Journal: Journal of hepatology

Article Title: TAZ-induced Cybb contributes to liver tumor formation in non-alcoholic steatohepatitis

doi: 10.1016/j.jhep.2021.11.031

Figure Lengend Snippet: (A) γH2AX and H2AX immunoblots from liver of mice injected with AAV8-H1-Scr or AAV8-H1-shTaz and fed the NASH diet for 16 weeks. (B) Percent 8-OHDG + HNF4α + cells in the livers of mice fed the NASH diet for 8 or 16 weeks (n = 5 mice/group; means ± SEM; **p < 0.01 by Student’s t-test). (C) Percent 8-OHDG + HNF4α + cells in livers of mice treated like those in panel A (n = 5 mice/group; means ± SEM; **p < 0.01 by Student’s t-test). (D) Ogg1, γH2AX, and H2AX immunoblots from AML12 cells transfected with GFP or Ogg1 plasmids and then incubated for 24 h with liposomes to deplete cholesterol (Lipo) and then 16 h with cholesterol-rich liposomes and palmitate (Lipo → Chol + Pal). (E-K) AAV8-TBG-Cre-treated Rosa NICD mice were fed the NASH diet and, 2 months later, injected with AAV8-TBG-GFP or AAV8-TBG-Ogg1. The mice were analyzed at month 4. (n = 7–8 mice/group; means ± SEM) (E) Experimental scheme. (F) Liver Ogg1 , Wwtr1 , Cybb and Nhej1 mRNA. (*p < 0.05 by two-way ANOVA/Sidak’s post-hoc analysis). (G) Immunoblots of the indicated proteins in the surrounding tumor tissue (ST) and tumor tissue from the livers of the two groups of mice. (H) Livers (arrows, tumors) and tumor numbers/mouse. (*p < 0.05 by Student’s t-test) (I) Percent Ki67 + HNF4α + cells in non-tumor tissue. (J) Liver sections were stained with H&E (upper images) and Sirius red (lower images), with quantification of inflammatory cells and percent Sirius red-positive area. Bars, 500 μm. (K) Percent TUNEL + cells in non-tumor areas.

Article Snippet: Male wild-type C57BL/6J mice (#000664, 9–10 weeks/old), Cybb fl/fl mice (#031777), and Rosa NICD mice (#008159) were from Jackson Laboratory (Bar Harbor, ME) and allowed to adapt in the animal facility for 1 week prior to random assignment to experimental cohorts.

Techniques: Western Blot, Injection, Transfection, Incubation, Liposomes, Staining, TUNEL Assay

Journal: Journal of hepatology

Article Title: TAZ-induced Cybb contributes to liver tumor formation in non-alcoholic steatohepatitis

doi: 10.1016/j.jhep.2021.11.031

Figure Lengend Snippet: (A-G) AAV8-TBG-Cre-treated Rosa NICD mice were fed the NASH diet and, 2 months later, injected with AAV8-H1-Scr or AAV8-H1-shCybb. The mice were analyzed at month 4. (A) Experimental scheme. (B) Nox2, γH2AX, and H2AX immunoblots from surrounding tissue (ST), with quantification (n = 3; means ± SEM; ***p<0.001, ****p<0.0001 by two-way ANOVA/Sidak’s post-hoc analysis). (C) Livers (arrows, tumors) and tumor numbers/mouse. (D) Liver sections were stained with H&E (upper images) and Sirius red (lower images) and quantified for the number of inflammatory cells and the percent Sirius red-positive area. Bars, 200 μm. (E) Percent TUNEL + cells in non-tumor areas. (F) Plasma ALT. (G) Average tumor diameter and percent Ki67 + HNF4α + cells in non-tumor areas. For C-G, n = 6–8 mice/group; means ± SEM; *p < 0.05 by Student’s t-test. (H-N) AAV8-TBG-Cre-treated Rosa NICD mice were fed the NASH diet and, 2 months later, injected with AAV8-H1-shTaz and either AAV8-TBG-LacZ or AAV8-TBG-Cybb. The mice were analyzed at month 4. (H) Experimental scheme. (I) Nox2, γH2AX, and H2AX immunoblots from ST, with quantification (n = 4; means ± SEM; **p<0.01, ***p<0.001 by two-way ANOVA/Sidak’s post-hoc analysis). (J) Livers (arrows, umors) and tumor numbers/mouse. (K) Liver sections were stained with H&E (upper images) and Sirius red (lower images), with quantification of inflammatory cells and percent Sirius red-positive area. Bars, 200 μm. (L) Percent TUNEL + cells in non-tumor areas. (M) Plasma ALT. (N) Average tumor diameter and percent Ki67 + HNF4α + cells. For J-N, n = 7 mice/group; means ± SEM; *p < 0.05 by Student’s t-test.

Article Snippet: Male wild-type C57BL/6J mice (#000664, 9–10 weeks/old), Cybb fl/fl mice (#031777), and Rosa NICD mice (#008159) were from Jackson Laboratory (Bar Harbor, ME) and allowed to adapt in the animal facility for 1 week prior to random assignment to experimental cohorts.

Techniques: Injection, Western Blot, Staining, TUNEL Assay, Clinical Proteomics