Journal: Frontiers in Cell and Developmental Biology

Article Title: Curculigoside Ameliorates Bone Loss by Influencing Mesenchymal Stem Cell Fate in Aging Mice

doi: 10.3389/fcell.2021.767006

Figure Lengend Snippet: CCG targeted TAZ to facilitate osteogenic differentiation of BMSCs in vitro . (A) MTT assays presented cell viabilities after CCG administration during osteogenesis of BMSCs. (B, C) Representative images of AR-S results in different mice groups. Scale bar: 100 μm. (D, E) Relative mRNA levels of TAZ, RUNX2, and OCN with or without CCG treatment. (F–I) TAZ, RUNX2, and OCN expression in protein levels with or without CCG treatment. * p < 0.05 vs. the control group. (J–L) SiTAZ plasmid significantly knocked down the mRNA and protein levels of TAZ during the osteogenesis of BMSCs. (M–Q) SiTAZ discounted the CCG induced up-regulation of RUNX2 and OCN. (R, S) Representative images of AR-S showed the differences of calcium deposits in different groups. Bar graphs showed the means ± SD from three independent experiments. Scale bar: 100 μm (n = 3) * p < 0.05 vs. the Vehicle group; # p < 0.05 vs. the Vehicle + CCG group.

Article Snippet: The designed and synthesized SiTAZ was used to knock down the TAZ expression (Genechem, China).

Techniques: In Vitro, Expressing, Control, Plasmid Preparation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Curculigoside Ameliorates Bone Loss by Influencing Mesenchymal Stem Cell Fate in Aging Mice

doi: 10.3389/fcell.2021.767006

Figure Lengend Snippet: CCG targeted TAZ to reduce adipogenic differentiation of BMSCs in vitro . (A) MTT assays presented cell viabilities after CCG administration during adipogenesis of BMSCs. (B, C) Representative images of Oil Red O staining reflected the lipid droplets. Scale bar: 100 μm. (D, E) Relative mRNA levels of TAZ, PPARγ, and perilipin to GAPDH were presented in Day 3 and Day 7 in the absence or presence of CCG during adipogenesis. (F–I) Relative protein levels of TAZ, PPARγ, and perilipin to GAPDH were presented in Day 3, in Day 7, and in Day 14 after the treatment. * p < 0.05 vs. the control group. (J–L) Relative mRNA and protein levels to GAPDH of TAZ were significantly knocked down by the SiTAZ plasmid during adipogenesis. (M–Q) SiTAZ discounted the CCG induced up-expression of TAZ and down-expression of PPARγ and perilipin at Day 3 after the treatment. (R, S) Representative images of Oil Red O staining showed the lipid droplets in different groups. Bar graphs showed the means ± SD from three independent experiments. Scale bar: 100 μm (n = 3) * p < 0.05 vs. the Vehicle group; # p < 0.05 vs. the Vehicle + CCG group.

Article Snippet: The designed and synthesized SiTAZ was used to knock down the TAZ expression (Genechem, China).

Techniques: In Vitro, Staining, Control, Plasmid Preparation, Expressing

Journal: Hepatology Communications

Article Title: A Therapeutic Silencing RNA Targeting Hepatocyte TAZ Prevents and Reverses Fibrosis in Nonalcoholic Steatohepatitis in Mice

doi: 10.1002/hep4.1405

Figure Lengend Snippet: Taz siRNA screen in mouse liver cells. (A) Forty‐eight siRNAs designed against mouse Taz ( Wwtr1 ) were transfected into mouse Hepa1‐6 cells using an siRNA concentration of 10 nM; Wwtr1 mRNA was assayed 24 hours later. Data were normalized to Gapdh , and the values in the graph are shown as percentage of Wwtr1 expression relative to the mean value of three nonspecific control siRNAs (= 100%). The average of four biological replicas ± SD is shown. (B) Dose‐response curves of siRNA6 and siRNA8; the average of four biological replicas ± SD is shown. (C) Hepa1‐6 cells were left untreated (−) or incubated under mock conditions with siCtrl or with 5 or 50 nM siTAZ‐1 or siTAZ‐2, which are the GalNAc conjugates of siRNA6 and siRNA8, respectively. Taz protein was analyzed by immunoblot and quantified by densitometry. (D) Primary mouse hepatocytes were incubated with the indicated concentrations of siTAZ‐1 and siTAZ‐2 and then assayed for Wwtr1 mRNA. The average of four biological replicas ± SD is shown. (E) Primary mouse hepatocytes, bone marrow‐derived macrophages, and HSCs were incubated with 100 nM siControl or siTAZ‐2 (shown as 2) and then assayed for Wwtr1 mRNA. The average of four biological replicas ± SEM is shown. (F) Human PBMCs were left untreated (−); incubated under mock conditions; with a negative control or three positive controls, as defined in Materials and Methods; with siCtrl; or with 100 nM siTAZ‐2. After 24 hours, IFN‐α2a and IP‐10 were assayed. Data are shown for PBMCs from three healthy donors as mean average of two to six biological replicas ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: C, siControl; Ctrl, control; HC, primary hepatocyte; HSC, hepatic stellate cell; Mac, bone marrow‐derived macrophage.

Article Snippet: Nontargeting GalNAc siRNA (siControl), siTAZ‐1, or siTAZ‐2 from Axolabs (Kulmbach, Germany) or phosphate‐buffered saline (PBS) were delivered as a single subcutaneous injection 9 weeks after the initiation of the NASH diet or as once‐weekly injections between 8 and16 weeks or 16 and 28 weeks of NASH‐diet feeding, using the doses indicated in the individual figures.

Techniques: Transfection, Concentration Assay, Expressing, Incubation, Western Blot, Derivative Assay, Negative Control

Journal: Hepatology Communications

Article Title: A Therapeutic Silencing RNA Targeting Hepatocyte TAZ Prevents and Reverses Fibrosis in Nonalcoholic Steatohepatitis in Mice

doi: 10.1002/hep4.1405

Figure Lengend Snippet: TAZ silencing using GalNAc‐siTAZ in NASH mice. (A) Experimental scheme. Male C57BL/6J mice were fed the NASH diet for 9 weeks and injected with PBS, GalNAc‐siControl, or GalNAc‐siTAZ‐1 or GalNAc‐siTAZ‐2 at the indicated doses. (B) Immunoblots of YAP and TAZ in the livers of the treated mice. (C,D) Immunoblots of TAZ and YAP in primary hepatocytes and NPCs isolated from mice treated with GalNAc‐siControl or GalNAc‐siTAZ‐2. Abbreviations: Ctrl, control; HC, primary hepatocyte.

Article Snippet: Nontargeting GalNAc siRNA (siControl), siTAZ‐1, or siTAZ‐2 from Axolabs (Kulmbach, Germany) or phosphate‐buffered saline (PBS) were delivered as a single subcutaneous injection 9 weeks after the initiation of the NASH diet or as once‐weekly injections between 8 and16 weeks or 16 and 28 weeks of NASH‐diet feeding, using the doses indicated in the individual figures.

Techniques: Injection, Western Blot, Isolation

Journal: Hepatology Communications

Article Title: A Therapeutic Silencing RNA Targeting Hepatocyte TAZ Prevents and Reverses Fibrosis in Nonalcoholic Steatohepatitis in Mice

doi: 10.1002/hep4.1405

Figure Lengend Snippet: Mice treated with GalNAc‐siTAZ during steatosis to NASH progression have markedly lower liver TAZ without affecting metabolic endpoints, liver triglyceride, or plasma lipids. (A) Experimental scheme. Male C57BL/6J mice were fed the NASH diet for 8 weeks and then injected once weekly for 8 additional weeks with PBS, GalNAc‐siControl, GalNAc‐siTAZ‐1, or GalNAc‐siTAZ‐2 at 10 mg/kg. (B,C) Immunoblots of liver TAZ in mice from the various experimental groups. (D) Assay of plasma ALT. For panel D, n = 8 mice/group, and values shown are mean ± SEM; * P < 0.05 using one‐way ANOVA. Abbreviations: 1, siTAZ‐1; 2, siTAZ‐2; C, siControl; P, phosphate‐buffered saline.

Article Snippet: Nontargeting GalNAc siRNA (siControl), siTAZ‐1, or siTAZ‐2 from Axolabs (Kulmbach, Germany) or phosphate‐buffered saline (PBS) were delivered as a single subcutaneous injection 9 weeks after the initiation of the NASH diet or as once‐weekly injections between 8 and16 weeks or 16 and 28 weeks of NASH‐diet feeding, using the doses indicated in the individual figures.

Techniques: Injection, Western Blot

Journal: Hepatology Communications

Article Title: A Therapeutic Silencing RNA Targeting Hepatocyte TAZ Prevents and Reverses Fibrosis in Nonalcoholic Steatohepatitis in Mice

doi: 10.1002/hep4.1405

Figure Lengend Snippet: Mice treated with GalNAc‐siTAZ during steatosis to NASH progression show improvements in liver inflammation and fibrosis and less liver cell death. (A) Livers of the mice described in Fig. were stained with H&E (upper row of images) and sirius red (lower row of images) and then quantified for inflammatory cells per field and percentage sirius red area. Scale bar, 200 μm; * indicates difference from P. (B‐E) The following endpoints were assayed in the livers of these mice: (B) indicated mRNAs; (C) percent of F4/80 + cells and α‐SMA + area; (D) MCP‐1 and TGF‐β1 concentration; and (E) percentage of liver cells that stained for TUNEL. For all panels, n = 8 mice per group; values shown are means ± SEM; * P < 0.05 using one‐way ANOVA. Abbreviations: 1, siTAZ‐1; 2, siTAZ‐2; C, siControl; P, phosphate‐buffered saline; Tnfa , tumor necrosis factor a mRNA.

Article Snippet: Nontargeting GalNAc siRNA (siControl), siTAZ‐1, or siTAZ‐2 from Axolabs (Kulmbach, Germany) or phosphate‐buffered saline (PBS) were delivered as a single subcutaneous injection 9 weeks after the initiation of the NASH diet or as once‐weekly injections between 8 and16 weeks or 16 and 28 weeks of NASH‐diet feeding, using the doses indicated in the individual figures.

Techniques: Staining, Concentration Assay, TUNEL Assay

Journal: Hepatology Communications

Article Title: A Therapeutic Silencing RNA Targeting Hepatocyte TAZ Prevents and Reverses Fibrosis in Nonalcoholic Steatohepatitis in Mice

doi: 10.1002/hep4.1405

Figure Lengend Snippet: GalNAc‐siTAZ treatment after the development of NASH reduces liver inflammation and fibrosis. (A) Experimental scheme. Male C57BL/6J mice were fed the NASH diet for 16 weeks and then injected once weekly for 12 additional weeks with PBS or GalNAc‐siTAZ‐2 at 10 mg/kg. (B) Livers were immunoblotted for TAZ and β‐actin and assayed for Wwtr1 mRNA. (C) Livers were stained with H&E (upper row of images) and sirius red (lower row of images) and then quantified for inflammatory cells per field and percentage sirius red area. Scale bars, 200 μm. (D) Livers were scored for fibrosis stage as described in Materials and Methods. P value was calculated from the average score for each group using the Wilcoxon rank‐sum test. (E‐K) The following endpoints were measured in the livers or plasma from these mice: (E) indicated mRNAs; (F) percentage of F4/80 + cells and the α‐SMA + area; (G) MCP‐1 and TGF‐β1 concentrations; (H) percentage of liver cells that stained for TUNEL; (I) plasma ALT; (J) Ihh mRNA; and (K) MMP activity. For all graphs, n = 7 (PBS) and n = 8 (GalNAc‐siTAZ‐2) mice. Values shown for all graphs except panel D are means ± SEM; * P < 0.05 using the two‐tailed Student t test. Abbreviations: 2, siTAZ‐2; Acta2, smooth muscle aortic α‐actin; AU, arbitrary unit; Dpt, dermatopontin; Opn, osteopontin; P, phosphate‐buffered saline.

Article Snippet: Nontargeting GalNAc siRNA (siControl), siTAZ‐1, or siTAZ‐2 from Axolabs (Kulmbach, Germany) or phosphate‐buffered saline (PBS) were delivered as a single subcutaneous injection 9 weeks after the initiation of the NASH diet or as once‐weekly injections between 8 and16 weeks or 16 and 28 weeks of NASH‐diet feeding, using the doses indicated in the individual figures.

Techniques: Injection, Staining, TUNEL Assay, Activity Assay, Two Tailed Test