PMC

(a–d) Digital image analysis quantification of collagen staining in control and β3 KO, β5 KO, β6 KO and itgb8^flox/flox;Pdgfrb-Cre (β8 Cre) mice (n = 6 male mice per group) after control (olive oil) or chronic CCl₄ treatment (x2 injections / week for 6 weeks). (e–h) Hydroxyproline analysis of liver tissue from control and β3 KO, β5 KO, β6 KO and itgb8^flox/flox;Pdgfrb-Cre mice. (i) Western blotting of integrin β8 subunit expression in control and itgb8^flox/flox;Pdgfrb-Cre HSCs culture activated for 7 days.

(a,b) Immunofluoresence micrographs of liver sections harvested from control (olive oil treated) or chronic CCl₄ treated (x2 injections / week for six weeks) mTmG;Pdgfrb-Cre reporter mice or Ai14;Pdgfrb-Cre reporter mice (n = 4 male mice per group). Scale bars, 100 μm. (c,d) Immunofluoresence micrographs of liver sections from control (olive oil treated) (c) or CCl₄ treated (d) Ai14;Pdgfrb-Cre mice (n = 4 male mice per group) stained for desmin, PDGFRβ or αSMA (green) with endogenous (not enhanced) Ai14-Td tomato report in red. (c) Scale bars, 25 μm. (d) Scale bar, 50 μm. Arrows indicate portal tracts in serial sections. (e) Gene expression profile of freshly sorted Td tomato positive cells from control (olive oil treated) or chronic CCl₄ treated Ai14;Pdgfrb-Cre mice (n = 4 male mice per group). (f) Immunofluoresence staining of Td tomato positive cells sorted from the uninjured livers of Ai14;Pdgfrb-Cre mice and plated on tissue culture plastic for 7 days. Left panel shows Ai14 reporter (red), DAPI (blue), middle panel shows αSMA (green), DAPI (blue), right panel shows merged image. Scale bar, 100μm. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test).

(a) qPCR (left panel) and western blot analysis of αSMA expression (right panel) in control and itgav^flox/flox;Pdgfrb-Cre (αv Cre) HSCs culture activated for 5 days. (b,c) qPCR analysis of Col1A1 and Col3A1 expression in control and itgav^flox/flox;Pdgfrb-Cre (αv Cre) HSCs culture activated for 5 days. (d) qPCR (left panel) and western blot analysis of αSMA expression (right panel) in control and itgav^flox/flox;Pdgfrb-Cre HSCs treated with 20 μg ml⁻¹ isotype control or αv integrin specific (clone RMV-7) antibodies for 5 days post plating. (e,f) qPCR analysis of Col1A1 and Col3A1 expression in control and itgav^flox/flox;Pdgfrb-Cre HSCs treated with 20 μg ml⁻¹ isotype control or αv integrin specific (clone RMV-7) antibodies for 5 days post plating. (g) qPCR analysis of Tgfb1 expression in control and itgav^flox/flox;Pdgfrb-Cre HSCs culture activated for 5 days. (h) TGF-β activation by control or itgav^flox/flox;Pdgfrb-Cre HSCs alone, or in the presence of TGF-β blocking antibody (clone 1D11, 40 μg ml⁻¹) or activated TGF-β (300 pg ml⁻¹). (i) Immunofluoresence micrographs of liver sections from control and itgav^flox/flox;Pdgfrb-Cre mice following chronic CCl₄ treatment (n = 8 male mice per group). Scale bars, 25 μm. (j) Digital image analysis quantification of phospho-SMAD3 staining. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test).

(a) Co-immunoprecipitation studies: Sorted Td tomato positive cells from uninjured livers of Ai14;Pdgfrb-Cre mice cultured for 7 days express αvβ1, αvβ3, αvβ5 and αvβ8. (b) Western blot and (c) qPCR analysis demonstrates induction of αv integrin expression during transition from the quiescent (day 0) to the culture activated phenotype (day 5,10) in wild type HSCs. (d) Western blotting of αv expression in control and itgav^flox/flox;Pdgfrb-Cre (αv Cre) HSCs culture activated for 5 days. (e) Picrosirius red staining (collagen deposition) (upper panels) and αSMA immunohistochemistry (lower panels) of liver tissue after olive oil or chronic CCl₄ treatment of control and itgav^flox/flox;Pdgfrb-Cre mice (n = 8 male mice per group). Scale bar, 200μm. (f) Digital image analysis quantification of collagen staining. (g) Hydroxyproline analysis. (h) Digital image analysis quantification of αSMA staining. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test).

(a) Dosing regime in the prophylactic liver fibrosis model (left panel). Alzet minipumps containing CWHM 12 or vehicle were inserted, followed by CCl₄ I.P. twice weekly for 6 weeks. Picrosirius red (upper) and αSMA immunohistochemistry (lower) of liver tissue from control and CWHM 12 treated mice (n = 6 female mice per group) after chronic CCl₄ treatment. Scale bar, 200μm. (b) Digital image analysis of picrosirius red staining. (c) Hydroxyproline analysis. (d) Digital image analysis of αSMA staining. (e) Dosing regime in the therapeutic liver fibrosis model (left panel). Mice were given CCl₄ I.P. twice weekly for 3 weeks, then Alzet minipumps containing either CWHM 12 or vehicle were inserted, followed by a further 3 weeks of CCl₄ I.P. twice weekly. Picrosirius red (upper) and αSMA immunohistochemistry (lower) of liver tissue from control and CWHM 12 treated mice (n =14 female mice per group) after chronic CCl₄ treatment. Scale bar, 200μm. (f) Digital image analysis of picrosirius red staining. (g) Hydroxyproline analysis. (h) Digital image analysis of αSMA staining. (i) Dosing regime in the therapeutic lung fibrosis model. Alzet minipumps containing either CWHM 12 or vehicle were inserted 14 days after treatment with bleomycin or saline, and lungs were harvested at 28 days. (j) Picrosirius red staining of lung tissue from control and CWHM 12 treated mice 28 days after bleomycin instillation (n = 15 female mice per group). Scale bar, 100μm (left panel). Hydroxyproline analysis (right panel). Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test).

(a) Immunofluoresence micrographs of lung sections from saline treated (left panel) or bleomycin treated (right panel) mTmG;Pdgfrb-Cre mice (n = 4 female mice per group) 28 days after instillation. Scale bar, 50 μm. (b,c) Immunofluoresence micrographs of lung sections from saline treated (b) or bleomycin treated (c) Ai14;Pdgfrb-Cre mice (n = 4 female mice per group). Scale bars, 25 μm. (d) Gene expression profile of freshly sorted Td tomato positive cells from saline treated (28 days post instillation) or bleomycin treated (14 or 28 days post instillation) Ai14;Pdgfrb-Cre mice (n = 4 female mice per group). (e) Picrosirius red staining of lung tissue 28 days after bleomycin instillation in control and itgav^flox/flox;Pdgfrb-Cre (αv Cre) mice (n = 12 female mice per group). Scale bar, 100μm. (f) Hydroxyproline analysis of lung tissue. (g) Immunofluoresence micrographs of left kidney sections from mTmG;Pdgfrb-Cre mice (n = 4 male mice per group) following sham operation or unilateral ureteric obstruction (UUO) for 14 days. Scale bar, 100 μm. (h) Gene expression profile of freshly sorted Td tomato positive cells from sham operated or UUO (day 7) Ai14;Pdgfrb-Cre mice (n = 4 male mice per group). (i) Picrosirius red staining of kidney tissue 14 days after UUO in control and itgav^flox/flox;Pdgfrb-Cre mice (n = 6 male mice per group). Scale bar, 100μm. (j) Digital image analysis of collagen staining. Data are mean ± s.e.m. *P < 0.05, **P < 0.01 (Student’s t test).