Journal: Cell Biology and Toxicology

Article Title: Investigating the role of Wnt3a and Wnt5a as critical factors of hepatic stellate cell activation in acute toxicant-induced liver injury

doi: 10.1007/s10565-024-09956-4

Figure Lengend Snippet: Primers used to detect mouse genes

Article Snippet: To measure accelerated HSC activation in vitro, mHSCs were cultured for 7 days and challenged with 10 ng/mL of recombinant mouse TGF –β (R&D Systems #7666-MB) as a positive control of HSC activation, or recombinant mouse Wnt3a (R&D Systems #1324-WN) or Wnt5a (R&D Systems #645-WN), DKK-1 (R&D Systems #5897-DK) on day 5.

Techniques:

Journal: Cell Biology and Toxicology

Article Title: Investigating the role of Wnt3a and Wnt5a as critical factors of hepatic stellate cell activation in acute toxicant-induced liver injury

doi: 10.1007/s10565-024-09956-4

Figure Lengend Snippet: Desmin-positive cells increase Fzd2/7 while proliferating hepatocytes increase Wnt3a and Wnt5a following acute liver injury. WT C57BL/6 J mice were challenged with CCl 4 or OO and tissues were harvested up to 72 h later. A ) Frozen liver sections were immunostained with Fzd2 or Fzd7 (green) or Desmin (red); images were quantified using Image J and co-localization of Fzd2 or Fzd7 and Desmin was determined by quantifying yellow pixels in Desmin-positive cells. B ) Integrated density of Wnt3a (red) or Wnt5a (red) expression in liver tissues. White boxes denote magnified regions demonstrating co-localized expression. N = 4–5 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: To measure accelerated HSC activation in vitro, mHSCs were cultured for 7 days and challenged with 10 ng/mL of recombinant mouse TGF –β (R&D Systems #7666-MB) as a positive control of HSC activation, or recombinant mouse Wnt3a (R&D Systems #1324-WN) or Wnt5a (R&D Systems #645-WN), DKK-1 (R&D Systems #5897-DK) on day 5.

Techniques: Expressing

Journal: Cell Biology and Toxicology

Article Title: Investigating the role of Wnt3a and Wnt5a as critical factors of hepatic stellate cell activation in acute toxicant-induced liver injury

doi: 10.1007/s10565-024-09956-4

Figure Lengend Snippet: Proinflammatory cytokine mediators are increased following acute liver injury and stimulate Wnt3a and Wnt5a production by hepatocytes and Kupffer cells. WT C57BL/6 J mice were challenged with CCl 4 or OO and tissues were harvested up to 72 h later. A ) Expression of proinflammatory marker mRNA ( tnfα, il-6, and il-10 ) was detected in mHSCs by qRT-PCR. B ) AML12 cells and C ) ImKCs were cultured in the presence or absence of tnf-α or il-6 for 24 h then fixed on glass coverslips and immunostained for Wnt3a and Wnt5a. Immunofluorescence was quantified using ImageJ; relative expression is denoted as arbitrary units of density. Data are from four independent experiments N = 3–4 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: To measure accelerated HSC activation in vitro, mHSCs were cultured for 7 days and challenged with 10 ng/mL of recombinant mouse TGF –β (R&D Systems #7666-MB) as a positive control of HSC activation, or recombinant mouse Wnt3a (R&D Systems #1324-WN) or Wnt5a (R&D Systems #645-WN), DKK-1 (R&D Systems #5897-DK) on day 5.

Techniques: Expressing, Marker, Quantitative RT-PCR, Cell Culture, Immunofluorescence

Journal: Cell Biology and Toxicology

Article Title: Investigating the role of Wnt3a and Wnt5a as critical factors of hepatic stellate cell activation in acute toxicant-induced liver injury

doi: 10.1007/s10565-024-09956-4

Figure Lengend Snippet: Spontaneous activation of mouse hepatic stellate cells is associated with increased expression of ECM genes, Wnt transducers, and β-catenin. A ) Primary mouse hepatic stellate cells were isolated from livers of Wild-type C57BL/6 J mice and cultured for up to 10 days. B ) Expression of activation marker mRNA ( col1a1 and α-sma ) was detected in mHSCs by qRT-PCR. C ) Expression of Wnt transducer mRNA ( fzd1, fzd2, fzd7 , and lrp6) was detected in mHSCs by qRT-PCR. Data are expressed as mHSCs cultured to day 7 and normalized to day 0.5. Data are significantly different from day 0.5, # P < 0.05. D ) mHSCs were fixed on glass coverslips and immunostained for β-catenin and Desmin. Immunofluorescence was quantified using ImageJ; relative expression is denoted as arbitrary units of density. E ) Expression of wnt3a and wnt5a mRNA was determined by qRT-PCR. Data are from three independent experiments, N = 5–6 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: To measure accelerated HSC activation in vitro, mHSCs were cultured for 7 days and challenged with 10 ng/mL of recombinant mouse TGF –β (R&D Systems #7666-MB) as a positive control of HSC activation, or recombinant mouse Wnt3a (R&D Systems #1324-WN) or Wnt5a (R&D Systems #645-WN), DKK-1 (R&D Systems #5897-DK) on day 5.

Techniques: Activation Assay, Expressing, Isolation, Cell Culture, Marker, Quantitative RT-PCR, Immunofluorescence

Journal: Cell Biology and Toxicology

Article Title: Investigating the role of Wnt3a and Wnt5a as critical factors of hepatic stellate cell activation in acute toxicant-induced liver injury

doi: 10.1007/s10565-024-09956-4

Figure Lengend Snippet: Exogenous Wnt3a and Wnt5a accelerate the spontaneous activation of mouse hepatic stellate cells. A ) Primary mouse hepatic stellate cells were isolated then cultured in the presence or absence of 10 ng/mL of exogenous TGF-β, Wnt3a, and Wnt5a from day 5 to day 7. B ) Expression of col1a1 mRNA was detected in mHSCs by qRT-PCR. C ) mHSC lysates were prepared and proteins separated by SDS-PAGE. α-SMA and HSC70 (loading control) were measured by Western blot. D ) β-catenin and Desmin expression was analyzed by immunofluorescence in cells following TGF-β, Wnt3a, and Wnt5a treatment. Red arrows highlight nuclear localization of β-catenin. E ) β-catenin expression and nuclear localization, and Desmin expression by immunofluorescence was quantified using ImageJ software. F ) Expression of axin2, ctgf, cyclind1, mmp9, mmp13, and timp1 mRNA and G ) wnt3a and wnt5a mRNA was detected by qRT-PCR. N = 5–6 per group. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: To measure accelerated HSC activation in vitro, mHSCs were cultured for 7 days and challenged with 10 ng/mL of recombinant mouse TGF –β (R&D Systems #7666-MB) as a positive control of HSC activation, or recombinant mouse Wnt3a (R&D Systems #1324-WN) or Wnt5a (R&D Systems #645-WN), DKK-1 (R&D Systems #5897-DK) on day 5.

Techniques: Activation Assay, Isolation, Cell Culture, Expressing, Quantitative RT-PCR, SDS Page, Control, Western Blot, Immunofluorescence, Software

Journal: Cell Biology and Toxicology

Article Title: Investigating the role of Wnt3a and Wnt5a as critical factors of hepatic stellate cell activation in acute toxicant-induced liver injury

doi: 10.1007/s10565-024-09956-4

Figure Lengend Snippet: HSC activation, via LRP6, occurs in a canonical Wnt-dependent manner. A ) Primary mouse HSCs were then cultured in the presence or absence of TGF-β, Wnt3a, and Wnt5a and the LRP-6 antagonist DKK-1 (10 ng/mL) from day 5 to day 7. B ) Expression of col1a1 mRNA was determined by qRT-PCR. C ) mHSC lysates were prepared and proteins separated by SDS-PAGE. α-SMA expression and HSC70 (loading control) were measured by Western Blot. α-SMA was normalized to HSC70 as a loading control and quantified using ImageJ software. D ) Expression of axin2, mmp9, and mmp13 mRNA; and E ) wnt3a and wnt5a mRNA was determined in mHSCs by qRT-PCR. Data are from three independent experiments. N = 3–6 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: To measure accelerated HSC activation in vitro, mHSCs were cultured for 7 days and challenged with 10 ng/mL of recombinant mouse TGF –β (R&D Systems #7666-MB) as a positive control of HSC activation, or recombinant mouse Wnt3a (R&D Systems #1324-WN) or Wnt5a (R&D Systems #645-WN), DKK-1 (R&D Systems #5897-DK) on day 5.

Techniques: Activation Assay, Cell Culture, Expressing, Quantitative RT-PCR, SDS Page, Control, Western Blot, Software

Journal: Cell Biology and Toxicology

Article Title: Investigating the role of Wnt3a and Wnt5a as critical factors of hepatic stellate cell activation in acute toxicant-induced liver injury

doi: 10.1007/s10565-024-09956-4

Figure Lengend Snippet: Mechanism of Wnt-induced HSC reprogramming during wound healing. Acute CCl 4 -induced liver injury damages pericentral hepatocytes and induces innate immune activation in the liver. Proinflammatory mediators, derived from immune cells, including TNF-α and IL-6, perform paracrine-mediated stimulation of resident parenchymal and non-parenchymal cells in the liver. Specifically, hepatocytes and Kupffer cells, in response to inflammatory stimuli, produce Wnt3a and Wnt5a in the microenvironment which in turn, signal to hepatic stellate cells (HSCs). Canonical Wnt-mediated β-catenin activation, signaling via Fzd 1, 2, and 7, drive fibrogenic gene expression leading HSC transdifferentiation to a myofibroblast phenotype to support liver regeneration following acute injury. WNT-; wingless; Fzd- frizzled receptor; LRP6 – low-density lipoprotein receptor-related protein 6; DKK-1 – dickkopf-1; col1a1- collagen 1; α-sma- smooth muscle actin alpha; ECM – extracellular matrix

Article Snippet: To measure accelerated HSC activation in vitro, mHSCs were cultured for 7 days and challenged with 10 ng/mL of recombinant mouse TGF –β (R&D Systems #7666-MB) as a positive control of HSC activation, or recombinant mouse Wnt3a (R&D Systems #1324-WN) or Wnt5a (R&D Systems #645-WN), DKK-1 (R&D Systems #5897-DK) on day 5.

Techniques: Activation Assay, Derivative Assay, Expressing

Journal: Molecular cell

Article Title: Members of an array of zinc finger proteins specify distinct Hox chromatin boundaries

doi: 10.1016/j.molcel.2024.08.007

Figure Lengend Snippet: Key Resources Table

Article Snippet: For caudalization of MNs, previously described methods were applied , . mESCs were differentiated into EBs in 2 days, and patterning was induced with 1 μM RA, 0.5 μM SAG, 75 ng/ml mouse WNT3A (R&D systems, Cat. 1324-WN-010/CF) and murine FGF-basic (Peprotech, Cat. 450–33) in a gradient of the described concentrations (50 ng/ml, 100 ng/ml, and 150 ng/ml) for 4 days.

Techniques: Virus, Recombinant, Magnetic Beads, SYBR Green Assay, Western Blot, Clone Assay, Plasmid Preparation, Software, Injection