Figure 3.

VCAM1-induced HSC activation is YAP1-dependent. Primary human HSCs were cultured using a cyto-soft plate with 0.2 kPa stiffness. hHSCs were treated with vechicle, 0.25 μM or 0.5 μM rhVCAM1 for 48 hours. (A) hHSCs activation was determined by mRNA levels of TIMP1 and PDGFRB. n=3. (B) Activation of hippo pathway protein YAP1 as well as its targets CTGF and ANKRD1 were examined by mRNA expression. n=3. (C) hHSCs were treated with rhVCAM1 for 48 hours. COL1A1 and phosphorylated and total YAP1 protein expressions were determined by western blotting. GAPDH was used as a loading control. The optical density of the bands (normalized to GAPDH for collagen 1 and to total YAP1 for phosphorylated YAP1) were quantified using ImageJ software and indicated below each band. (D) hHSCs were treated with vechicle or rhVCAM1 0.5 μM for 5 days, YAP1 subcellular locolization was examined by confocal microscopy and immunofluorescence using an anti-YAP1 antibody (red), HSCs activation was accessed by aSMA immunofluorescence (green). YAP1 positive nuclei were quantified from 5 random fields using imageJ software. Scale bar: 10μm. **, ***, **** indicate statistical significance with p < 0.01, p < 0.001 and p < 0.0001, respectively.