Imr 90

IMR-90 (product #CCL-186) was purchased from ATCC, Inc. (USA). Vybrant^® CFDA SE Cell Tracer Kit (product #V12883, excitation/emission maxima: 492/517 nm) was purchased from Life technologies, Inc. (USA). To prepare labeled live IMR-90 cell samples, IMR-90 cells were grown inside a petri dish (Corning, USA) filled with the culture medium DMEM (life technologies, USA) supplemented with 10% FBS (life technologies, USA) to induce an adherent state. 2.5μL 10mM CFDA SE solution was added into 5mL DPBS (life technologies, USA) for dilution. When the cells reached the desired density, the culture medium was removed from the dish and the aforementioned DPBS with the probe was added inside. After incubating the cells for 15 minutes at 37 °C, the loading solution was replaced with fresh, pre-warmed culture medium and cells were incubated for another 30 minutes at 37 °C. Finally, the old medium was again replaced with fresh and pre-warmed medium for washing.

Primary human lung microvascular endothelial cells (HMVEC-L) were purchased from Lonza (CC-2527). HMVEC-L were cultured in EGM^™-2MV Microvascular Endothelial Cell Growth Medium-2 BulletKit^™ (Lonza, CC-3202) at 37°C, 14% O₂, 5% CO₂. Human lung fibroblasts IMR-90 were purchased from Coriell Institute (I90). IMR-90 cells were cultured in DMEM (Corning, 01–017-CV) supplemented with 10% FBS (R&D Systems, S11550H), 100 units/mL penicillin, and 100 μg/mL streptomycin (R&D Systems, B21210) at 37°C, 3% O₂, 10% CO₂. For all experiments performed, both HMVEC-L and IMR-90 were cultured in 96-well microplates appropriate for microscopy imaging (Corning, 3904), with media changes every 2–3 days.
To achieve serum starvation in HMVEC-L (Fig. 3c), cells were washed twice in DPBS containing Ca²⁺ and Mg²⁺ (Gibco, 14040–117) and then cultured for 72 h in low serum EGM^™-2MV medium (0.5% FBS instead of 5.0% FBS). To achieve high confluency conditions (Fig. 3e), cells were seeded at high density (25,000 cells/cm²) and further cultured for 7 days before irradiation; non-senescent control cells were also seeded at the same density and cultured for 7 days before analysis.