Journal: PLOS ONE

Article Title: Activated Gab1 drives hepatocyte proliferation and anti-apoptosis in liver fibrosis via potential involvement of the HGF/c-Met signaling axis

doi: 10.1371/journal.pone.0306345

Figure Lengend Snippet: Humanized-liver mice were injected into the tail vein with 1 × 10 4 and 1 × 10 5 FFU HCV in 100 μL PBS. Liver tissues were harvested 7 days post infection (dpi) and hepatocytes were isolated. Lysates were prepared from isolated hepatocytes and used for reverse-phase protein microarray analysis (RPMA). (A) Schematic diagram showing the RPMA process. (B, C) Heatmap showing the protein fold change in isolated hepatocytes from HCV-infected humanized liver mouse. Data are expressed as mean ± SD (n = 3). * P < 0.05, ** P < 0.01 and *** P < 0.001 were considered statistically significant, as assessed using a t-test.

Article Snippet: Human hepatocytes repopulated fah(-/-) rag1(-/-) IL2rgnull on NOD (FRGN) mice were provided by Yecuris (Tualatin, OR, USA).

Techniques: Injection, Infection, Isolation, Microarray

Journal: PLOS ONE

Article Title: Activated Gab1 drives hepatocyte proliferation and anti-apoptosis in liver fibrosis via potential involvement of the HGF/c-Met signaling axis

doi: 10.1371/journal.pone.0306345

Figure Lengend Snippet: Humanized-liver mice were injected into the tail vein with 1 ×10 4 and 1 × 10 5 FFU of HCV in 100 μL PBS. Liver tissues were harvested 7 dpi and hepatocytes were isolated. (A) mRNA was extracted using TRIzol, and mRNA expression levels were analyzed by qRT-PCR. HPRT was used as an endogenous control for normalization in cells. (B) mRNA expression of TGF-β was analyzed by qRT-PCR. Data are expressed as mean ± SD (n = 3). (C) The expression of caspase-3 ( P = 0.31) and caspase-9 ( P = 0.0001) was analyzed by RPMA. * P < 0.05, ** P < 0.01 and *** P < 0.001 were considered statistically significant, as assessed using a t-test.

Article Snippet: Human hepatocytes repopulated fah(-/-) rag1(-/-) IL2rgnull on NOD (FRGN) mice were provided by Yecuris (Tualatin, OR, USA).

Techniques: Injection, Isolation, Expressing, Quantitative RT-PCR, Control

Journal: PLOS ONE

Article Title: Activated Gab1 drives hepatocyte proliferation and anti-apoptosis in liver fibrosis via potential involvement of the HGF/c-Met signaling axis

doi: 10.1371/journal.pone.0306345

Figure Lengend Snippet: Hepa1-6 cells were treated with 20 mM of CCL4 and 20 mg/mL of mouse hepatocytes growth factor (mHGF) for 48 hrs and 30 min respectively. Cell lysates and culture supernatants were collected and analyzed. (A) mRNA expression of Gab1 was measured by qRT-PCR. (B) Protein levels of pGab1 and GAPDH were analyzed by western blot. (C) Intracellular pGab1 (Y627) protein levels were analyzed by confocal microscopy. Confocal microscopic images of pGab1 (Y627) (green) expression in Hepa1-6 cells are shown. Nuclei were stained with DAPI. Scale bar, 20 μm. (D) mRNA expression levels of mTOR, STAT3, c-Raf, MET, ERK1, and ERK2 were measured by qRT-PCR. Data are expressed as mean ± SD (n = 3). * P < 0.05 and ** P < 0.01 were considered statistically significant, as assessed using a t-test.

Article Snippet: Human hepatocytes repopulated fah(-/-) rag1(-/-) IL2rgnull on NOD (FRGN) mice were provided by Yecuris (Tualatin, OR, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Confocal Microscopy, Staining

Journal: PLOS ONE

Article Title: Activated Gab1 drives hepatocyte proliferation and anti-apoptosis in liver fibrosis via potential involvement of the HGF/c-Met signaling axis

doi: 10.1371/journal.pone.0306345

Figure Lengend Snippet: Hepa1-6 cells were treated with carbon tetrachloride (CCL4) and transfected with either AllStars Negative Control siRNA (5 nM) (Qiagen) or Gab1 siRNA (20 pM) (Sigma) using Lipofectamine™ 2000 Transfection Reagent (Thermo Fisher Scientific), following the manufacturer’s protocol. Mouse hepatocytes growth factor (mHGF) was added 2 days post transfection for 30 min, and cell lysates were used for analysis. (A) mRNA expression level of Gab1 was measured by qRT-PCR (n = 4). (B) Protein levels of pGab1, Gab1 and GAPDH were assessed by western blot analysis. (C) Protein levels of pSTAT3, pERK1/2, GAPDH, pro-caspase-3, and cleaved caspase-3 were assessed by western blot analysis. (D) mRNA expression of TGF-β is presented (n = 3). (E) Schematic diagram of the possible role of Gab1 in liver disease. During the early phase of liver damage, the production of TGF-β activates TGF-β signaling, leading to cell growth inhibition and apoptosis. Subsequently, damaged hepatocytes activate cell proliferation pathway and develop chronic liver disease such as fibrosis via pGab1 activation of the PI3K/AKT and ERK pathways. ** P < 0.01 was considered statistically significant, as assessed using a t-test.

Article Snippet: Human hepatocytes repopulated fah(-/-) rag1(-/-) IL2rgnull on NOD (FRGN) mice were provided by Yecuris (Tualatin, OR, USA).

Techniques: Transfection, Negative Control, Expressing, Quantitative RT-PCR, Western Blot, Inhibition, Activation Assay