Table 1.
Human-specific antibody sets for reverse-phase protein microarray (RPMA).
Table 2.
Human and mouse-specific primer sets for RT-qPCR.
Fig 1.
Early HCV infection enhanced cell survival signaling pathways associated with TGF-β in hepatocytes while phosphorylated Gab1 decreased.
Humanized-liver mice were injected into the tail vein with 1 × 104 and 1 × 105 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.
Fig 2.
pGab1 expression levels are inversely related to TGF-β synthesis during acute viral infection of hepatocytes.
Humanized-liver mice were injected into the tail vein with 1 ×104 and 1 × 105 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.
Fig 3.
pGab1 levels decreased over time in HCV-infected Huh7.5.1 cells.
Huh7.5.1 cells were infected with HCV at 0.5 MOI. Cell lysates were collected daily for 5 days for analysis. (A) mRNA was extracted using TRIzol and mRNA expression of Gab1 was analyzed by qRT-PCR. HPRT was used as an endogenous control for normalization in cells. (B) Intracellular pGab1 (Y627) protein levels were assessed by confocal microscopy. Confocal microscopic images of HCV core (red) and pGab1 (Y627) (green) expression in Huh7.5.1 cells for 3 dpi are presented. Nuclei were stained with DAPI. Scale bar, 20 μm. (C) Quantification of the pGab1 and HCV positive areas per Huh7.5.1 cell for either uninfected or HCV-infected cells for D1, D2, and D3. Using ImageJ, the area and the count for pGab1 and HCV core protein in the cells were quantified. (D) mRNA expression of mTOR, STAT3, c-Raf, MET, and ERK2 were measured by qRT-PCR. (E) Protein levels of pSTAT3, STAT3, pERK1/2 and GAPDH were assessed by western blot analysis. (F) mRNA expression of TGF-β was analyzed by qRT-PCR, and TGF-β protein levels were measured by ELISA using cell culture supernatant. 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.
Fig 4.
pGab1(Y627) levels are elevated during the development of liver fibrosis.
(A) Mice were administered with CCL4 every 3 days orally, receiving escalating doses over time. Liver tissues were collected for analysis 6 weeks after CCL4 induction of liver fibrosis. (B) Hydroxyproline level was analyzed using wet liver tissues (30–50 mg). mRNA was extracted using TRIzol, and mRNA expression was analyzed by qRT-PCR. HPRT was used as an endogenous control for normalization in cells. (C) Liver tissue was collected and fixed in 10% buffered formalin for 24 hours. Serial 5-μm paraffin sections were prepared for staining with H&E and picosirius red. Quantification of red-stained collagen in an image of a mouse liver tissue section stained with Sirius Red using the ImageJ program is shown. To obtain statistical results, we repeated the quantification five times in different areas of liver tissue sections (n = 15). (D and E) mRNA and protein expression levels of Gab1 and pGab1 (Y627) were analyzed by qRT-PCR and western blot respectively. (F) Protein expression of Ki67, pSTAT3, CYP2E1, pERK1/2, and GAPDH was analyzed by western blot. (G) mRNA expression levels of TGF-β were measured by qRT-PCR (n = 3). (H) mRNA expression levels of mTOR, STAT3, c-Raf, MET, and ERK2 were measured by qRT-PCR (n = 6). *P < 0.05, ** P < 0.01 and *** P < 0.001 were considered statistically significant, as assessed using a t-test.
Fig 5.
Gab1 expression increases in mouse hepatoma cell line treated with CCL4 and HGF.
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.
Fig 6.
Gab1 gene silencing reduces cell proliferation and induces cell death signal in mouse hepatoma cell line treated with CCL4 and HGF.
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.