Figure 3: HSCs are the principal source of myofibroblasts in CCl₄-induced liver fibrosis.

(a) Representative fluorescent images of whole livers from untreated and CCl₄-treated mice (n=3) show LratCre-labelled ZsGreen-positive macroscopic fibrotic septa in CCl₄-treated liver. (b). Frozen liver sections from CCl₄-treated LratCre-positive mice were stained with desmin (upper panel) or αSMA (middle panel) to demonstrate co-localization of HSC marker desmin or αSMA and LratCre-induced ZsGreen by confocal microscopy. Confocal microscopy was employed to show co-localization of Col-GFP reporter, marking activated myofibroblasts and LratCre-induced tdTomato expression (lower panel). Quantification of αSMA-expressing cells that are derived from LratCre-labelled ZsGreen-positive HSCs in fibrosis induced by 9 × CCl₄ (n=4, upper graph) or Col-GFP-expressing cells that are derived from LratCre-labelled tdTomato-positive HSCs in fibrosis induced by 9 × CCl₄ treatment (n=4, lower graph). (c) Co-localization of αSMA with tdTomato and Col-GFP in 9 × CCl₄-induced liver fibrosis was determined by confocal microscopy employing far-red secondary antibody for αSMA detection. (d,e) LratCre mice, expressing Cre-inducible diphteria toxin receptor (iDTR) received either vehicle (n=4) or dipheria toxin (DT, n=4) during CCl₄-induced liver fibrosis induction. Expression of αSMA and desmin was determined by immunohistochemstry and quantified (d), expression of fibrogenic genes was determined by qPCR (e). Scale bars, 1 mm (a), 100 μm (b,c) and 200 μm (d). Data are shown as means±s.d. *P<0.05, **P<0.01 (determined by Student’s t-test).