Figure 3. The secretome of senescent fibroblasts is profibrotic.

IMR90 lung fibroblasts were exposed to irradiation (10 Gy). Twenty-one days later, senescence was confirmed by (a) SA-β-gal staining (scale bar, 100 μm) and (b) RT–PCR assessment of p16, p21, MCP1 and IL6 expression relative to TBP levels in 10 Gy exposed (red) and sham-treated (grey) cells (mean±s.e.m.; n=3, *P≤0.05), as well as (c) immunoanalysis of secreted SASP components within 10 Gy-exposed CM (SASP-CM), relative to CCM (mean±s.e.m.; n=4, **P<0.01, *P≤0.05, ^¥P=0.08 and ^#P≤0.1.) (d) IMR90 cells were treated with media collected 21 days post 10 Gy, -sham exposure or control as follows: NCM (black), NCM+2 ng ml⁻¹ TGFβ (orange), CCM (grey) or SASP-CM (red). IMR90 cells were treated with the indicated media for 72 h then immunostained for αSMA (green) and DAPI (blue). Percentage αSMA-positive cells were determined blindly, using a visual threshold (mean±s.e.m.; n=2–4 independent experiments; **P<0.01 and *P≤0.05). (e) IMR90 cells were plated onto 6.4 kPA matrices for traction force microscopy in the presence of the indicated media for 72 h. Representative traction heat maps, root mean square (RMS) traction and peak traction are depicted (mean±s.e.m.; n=2–4 independent experiments with a minimum of 20 independent cells per condition; **P<0.01 and *P≤0.05 versus control CM). (f) IMR90 cells were treated with indicated media for 72 h before RNA isolation. RT–PCR expression of ACTA2, COL1A1, COL1A2 and FN1 were measured relative to GAPDH levels (mean±s.e.m.; n=2–4 independent experiments, *P≤0.05). (g) Human primary lung fibroblasts were exposed to irradiation (10 Gy). Twenty days post irradiation, cells were treated with the indicated concentrations of DQ (yellow) or navitoclax (green). Cell viability 3 days after drug treatment was measured by ATPLite assays and is indicated as a percentage of plating density at day 0 of treatment. (mean±s.e.m.; n=4, t-test, ***P<0.001, **P<0.01 and *P≤0.05).