Fig 2: YAP-1 and MYL9 mediate pro-fibrotic aspects of liver fibrosis in activated HSCs.(a,b) Quantified increase in total MYL9 and YAP-1 protein levels on activation of rat HSCs from n=3 experiments (a) with representative immunoblot shown in b. (c–e) Total YAP-1 and MYL9 are diminished in activated mouse HSCs (‘Control’) following integrin beta-1 loss (‘Itgb1-null’). Quantification from n=3 experiments shown in c with representative immunoblot in d. In the immunofluorescence in e, note the rounded inactivated appearance of the Itgb1-null cells. The remaining total YAP-1 signal is more cytoplasmic (see h,i). Scale bar, 50 µm. (f,g) Itga11 knockdown in activated rat HSCs using two different siRNA oligos. Data for each oligo are shown relative to its own scrambled control in either black or grey (n=3 for each). (f) Detection of Myl9 transcripts was diminished to an almost identical extent as for Itga11. (g) Protein detection of MYL9 and total YAP-1 was also diminished following Itga11 knockdown. (h,i) The proportion of phosphorylated YAP (PYAP, inactive form), is increased following integrin beta-1 loss (‘Itgb1-null’) from activated mouse HSCs (‘Control’; n=3 experiments) and localises more predominantly to the cytoplasm (i). DAPI, blue nuclear counterstain, is shown. Scale bar, 50 µm. (j) Luciferase activity (in relative light units; RLU) following co-transfection of constructs containing the wild-type (MYL9-TEAD) or mutated (MYL9-?TEAD) TEAD motif from the 3'-untranslated region of the MYL9 gene with empty vector (Control) or YAP expression vector. Results are normalized to a Renilla vector and expressed relative to the control MYL9 luciferase construct without YAP. (k) Transcript levels by qRT–PCR following inhibition of YAP-TEAD interaction using VP in activated rat HSCs expressed relative to DMSO control. Two-tailed unpaired t-test was used for statistical analysis. Data are shown as means±s.e.m. *P<0.05, **P<0.01, †P<0.005, ‡P<0.001.