Figure 1.

TGF-β1 (transforming growth factor-β1) activated the p16-pRb pathway and induced senescence as well as secretion of profibrotic mediators in primary mouse alveolar type II (ATII) cells. Freshly isolated mouse ATII cells were treated with 2 ng/ml of TGF-β1 in serum-free medium for 48 hours and then incubated with TGF-β1–free and serum-free medium for an additional 24 hours. (A and B) Senescence-associated β-galactosidase (SA-β-gal) activity was revealed by 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-gal) staining. (C and D) Western analyses of the proteins of interest in isolated ATII cells. Representative Western blots (C). Quantification of the band intensities, normalized by GAPDH (D). (E) Caspase 3/7 activity in the conditioned medium (CM) was measured with a kit from Promega. (F–K) The amounts of different cytokines, chemokines, and growth factors in the CM were assessed by ELISA. *Significantly different from untreated ATII cells (P < 0.05; n = 3–6). C-casp3 = cleaved caspase 3; IGFBP3 = insulin-like growth factor–binding protein 3; PAI-1 = plasminogen activator inhibitor-1; PDGF = platelet-derived growth factor; pRB = phosphorylated retinoblastoma protein; RLU = relative luminescence units.