Figure 4
From: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis
TGF-β1 associates with areas of decreased clusterin expression in fibrotic lung and down-regulates fibroblast clusterin mRNA and protein expression in vitro. Serial sections prepared from IPF lung (n = 3) were stained immunohistochemically to localize TGF-β1 staining ((A), red/brown, nuclei blue) and clusterin ((B), red/brown, nuclei blue) in fibroblastic foci. (A,B) Representative images of immunohistochemical staining suggest that TGF-β1 localizes to ECM, fibroblasts and macrophages, whilst staining for clusterin is weak or undetectable. (C) In vitro analysis of clusterin mRNA levels in TGF-β1 stimulated (40 pM) human lung fibroblasts was performed via qRT-PCR and shows a time-dependent down-regulation of clusterin expression that was maximal at 24–48 h (n = 3) in response to TGF-β1. Clusterin protein levels were also down-regulated in response to TGF-β1 (40 pM) compared to control at 24 h and 48 h as demonstrated by western blotting at 24 h (D) and immunofluorescent staining at 48 h (E, red, nuclei - blue). (F) Semi-quantitative analysis of fluorescent signal of panel E: clusterin signal (pixel intensity) was normalized to cell numbers per visual field and compared to control (n = 6). Full-length western blots are presented in Supplementary Figure e4. Different cell populations/structures are indicated by arrows; f - fibroblast-like cell, m - macrophage, he - hyperplastic epithelial cell, ecm – extracellular matrix. *P < 0.05, **P < 0.01; scale bar 100 µm (A) and 10 µm (E).
