Research Paper Volume 15, Issue 14 pp 6641—6657

Figure 4. The secretome of senescent human lung fibroblasts as mediator of murine lung fibrosis. (A) Diagram showing cytokine concentrations in conditioned media (CM) corresponding to 0.5 million cells collected from irradiated SEN-IMR90 (SEN-CM) compared with proliferating IMR90-derived CM (NS-CM) as control, were quantified by using human cytokine arrays; n = 4 each group, independent experiments. Statistical significance was assessed by the two-tailed Student’s test: ^*p < 0.05. (B) Mouse Embryo Fibroblasts (MEF) incubated with SEN-CM or NS-CM as control for 6 days. Senescence was confirmed by SABG staining (scale bar, 100 μm). (C) Transcriptional upregulation of the profibrotic secretome components (IL-6, Tgf-β and Col1a2) was confirmed by RT-PCR at 2 days post-exposure to the indicated CM; n = 6 SEN-CM and n = 3 NS-CM group, independent experiments. Statistical significance was assessed by the two-tailed Student’s test: ^*p < 0.05. (D) Representative immunofluorescence images showing double staining of Cdkn1a/p21^Cip1/Waf1 (red), and α-smooth muscle actin (α-SMA) (green) (10×, scale bar, 100 μm). (E) Quantification of the average number of α-SMA/p21 double positive cells as observed in the images shown in panel D using ImageJ. Quantification was performed from 4 experiments with >25 cells quantified for each condition. Statistical significance was assessed by the two-tailed Student’s t-test: ^*p < 0.05. (F) Diagram showing the experimental plan to evaluate the effect of the secretome in nude mice lung. These animals were intratracheally delivered SEN-CM or NS-CM, normalized by the number of cells (corresponding to 5 x 10⁵ cells each group), using PBS as negative control; n = 10 each group. (G) Hydroxyproline content in the right lung tissues of mice injected with SEN-CM or NS-CM, compared with control; n = 10 each group. (H) Representative images of lung sections of nude mice 21 days after instillation of SEN-CM or NS-CM, or PBS as negative control, stained with Hematoxylin Eosin (HE) and Masson’s Trichrome (MT) (40×, scale bar 100 μm) showing that SEN-CM initiated a cascade of the events that induced mild fibrosis. Statistical significance was assessed by the one-way ANOVA with Tukey test: ^***p < 0.001; ^*p < 0.05. For further explanations, see text.