Journal: Scientific Reports
Article Title: Proline-rich tyrosine kinase 2 mediates transforming growth factor-beta-induced hepatic stellate cell activation and liver fibrosis
Figure Lengend Snippet: Profibrogenic TGF-β1 activates YAP in PYK2-dependent manner in vivo and in vitro. ( A ) qPCR of YAP target genes expression in activated mHSC that were pretreated with DMSO or Verteporfin before TGF-β1 stimuli, and shown as fold change (n = 3). ( B ) qPCR of YAP target genes and SMAD7 mRNA expression (shown as fold change, n = 3) and western blot of YAP and TAZ (n = 2) in LX2 transfected with siRNA of control, YAP, or TAZ, followed by TGF-β1 stimuli. ( C ) YAP reporter assay in LX2 transfected with either IFN-luc or YAP-luc (8xGTIIC-luc) followed by TGF-β1 stimuli as indicated (n = 3). ( D ) qPCR of CTGF and CYR61 mRNA expression in LX2 that were transfected with either vector alone or YAP(wt) and then followed by the treatments as indicated (n = 3). ( E ) YAP reporter assay in LX2 transfected with siRNA of either control or PYK2, followed by 2nd transfection with vector alone or YAP(wt) (n = 2). ( F ) IHC co-staining of α-SMA and YAP in paraffin liver sections of CCl 4 -treated mouse (n = 4). Arrows indicate the co-localized regions. ( G ) Western of YAP expression in primary mHSCs at the indicated times on in vitro culture. ( H ) IHC of YAP in CCl 4 -treated mouse livers (n = 3) and western blot of YAP expression in livers from O + V (n = 3), O + CCl 4 (n = 3), CCl 4 + PF (n = 3). ( I ) IHC of α-SMA, p-PYK2(402), and YAP expression in serial liver sections from patients with cirrhosis (n = 4). The enlarged insets were shown below. The luciferase activities were normalized with co-transfected renilla luciferase activity. Scale bar: 200 μm in ( F , H ), 500 μm in ( I ), and 200 μm in insets of ( I ), Student's t-test; * p
Article Snippet: To activate TGF-β1 signaling in vitro, serum-starved cells were stimulated by 5 ng/ml recombinant human TGF-β1 protein (R & D Systems) for 1.5 h in most experiments, with the exception that 10 h or overnight treatment was required to induce expression of α-SMA and type I collagen.
Techniques: In Vivo, In Vitro, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Reporter Assay, Plasmid Preparation, Immunohistochemistry, Staining, Luciferase, Activity Assay