Figure 3.

Lung fibrosis induced by senescent human lung fibroblasts is progressive. (A) Diagram showing the experimental plan to evaluate the dynamics of the development of pulmonary fibrosis in nude mice, as well as representative images of lung sections stained with Masson’s Trichrome (MT) (20×, scale bar 100 μm). These animals received intratracheal instillation of irradiated SEN-IMR90, compared with PBS-exposed mice at early endpoints, and bleomycin-challenged mice (single injection, dose of 3 UI/kg) at late endpoints; n = 3 each group. (B) Hydroxyproline content in the right lung of mice injected with SEN-IMR90 compared with control (PBS). (C) Relative expression of the mRNA coding for murine Col1a2 in the lungs of the same mice as in panel B. (D) Relative expression of the mRNA coding for murine Cdkn1a/p21^Cip1/Waf1 in the same mice as in panel B. For panels B, C and D, n = 3 for each experimental group and statistical significance was assessed by the one-way ANOVA with Tukey test: ^**p < 0.01; ^*p < 0.05. For panels B, C and D, the group labelled SEN-IMR90 (2 months) is the same group labelled SEN-IMR90 in Supplementary Figure 1A–1C, and the data are the same. (E) Images of lung sections showing positive cells using IHC staining for HuNu and mouse p21 (Cdkn1a/p21^Cip1/Waf1) from mice injected with SEN-IMR90 or control. Engraftment of senescent cells (arrows) in mice sacrificed after different time points (3 hours, 48 hours, and 1 month), showing their dramatic reduction after 48 hours post-transplantation, and the gradually increase of mouse p21 over time (20×, scale bar 100 μm).