Figure 5.

Myofibroblastic localization of Gli1 and the effect of hedgehog inhibition on TGFβ-activated human HSCs in vitro and human liver slices ex vivo. (a) Gene expression of hedgehog pathway-related genes (Shh, Ptch1, Smo, Gli1, Gli2 and Sox9) in ITGA11-KD HSCs versus control HSCs, n=3. **P<0.01 represents significance versus control HSCs. (b) Gene expression of hedgehog pathway-related genes (Sox9 and Gli1) in TGFβ-treated ITGA11-KD HSCs versus TGFβ-treated control HSCs, n=4. ^#P<0.05 and ^##P<0.01 versus control HSCs (dashed line); *P<0.05 and **P<0.01 versus TGFβ-treated HSCs. (c) Gene expression of ITGA11 in control HSCs and ITGA11-KD HSCs with or without Shh (5 μg ml⁻¹), n=3. ^#P<0.01 versus Shh-treated HSCs, **P<0.01 versus control HSCs. (d) α-SMA and Gli1 co-immuno-stained human liver sections. Gli1 (red, first column), α-SMA (green, second column) and merged image (third column). Fourth column shows the magnified image depicting co-localization. Nuclei are stained blue with DAPI. (e) Collagen-I- and vimentin-stained HSCs treated with or without TGFβ (5 ng ml⁻¹)±10 μM of the hedgehog inhibitor (LDE225). Gene expression of fibrotic parameters collagen-I, α-SMA and vimentin in HSCs treated with medium alone, TGFβ (5 ng ml⁻¹)±10 μM LDE225, n=3. ^#P<0.05, ^##P<0.01 versus the control cells; *P<0.05 versus TGFβ-treated cells. (f) Graph depicts % 3D collagen-I gel contraction after 24, 48 and 72 h of treatment with or without TGFβ (5 ng ml⁻¹)±10 μM LDE225, n=3. ^#P<0.05, ^##P<0.01 versus control cells; *P<0.05 versus TGFβ-treated cells. Gene expression of Acta2, TIMP1, PDGFβR and Collagen-I (g) and ITGA11, Sox9, Gli1 and ITGA5 (h) in the slices obtained from fibrotic livers from human patients incubated with medium (control) or 10 or 15 μM LDE225. n=3 patients (3 slices each). *P<0.05 and **P<0.01 versus control group. Control cells were transfected with control/scrambled shRNA plasmid. ITGA11-KD cells were transfected with the ITGA11 shRNA plasmid.