Elisas

mRNA expression was analyzed by real-time reverse transcription (RT) PCR in accordance with the previous study [37 (link)]. The relative expression was calculated as ddCt and data were normalized to the expression of β-actin mRNA for each sample. Table S2 shows the primer sequences for each gene.
Standard Western blotting techniques were used.The following antibodies were used:β-actin(Cell Signaling Technology, Danvers, MA, USA), LC3B(Cell Signaling Technology, Danvers, MA, USA), ATG12(Cell Signaling Technology, Danvers, MA, USA), Snail(Cell Signaling Technology, Danvers, MA, USA), E-cadherin (Cell Signaling Technology, Danvers, MA, USA), NOX4(Santa Cruz Biotechnology, Dallas, TX, USA), p62(Santa Cruz Biotechnology, Dallas, TX, USA), Poldip2(Abcam, Cambridge, UK), and ZO-1 (Abcam, Cambridge, UK). The presence of transforming growth factor (TGF)-β, interleukin (IL)-6, and tumor necrosis factor (TNF)-α in mouse serum and cell conditional mediumwas quantified using enzyme-linked immunosorbent assays (ELISAs; Cusabio, Wuhan, China).

The supernatant of hAECs cocultivated with TNIL-exos, TL-exos and A549-exos for 24 h was centrifuged at 1000×g for 15 min before use and stored at −80°C. Concentrations of the SASP markers MMP9, GM-CSF, TNF-a, IL-6 and IL-8 were measured by ELISAs (Cusabio Biotech). The experimental process was performed following the manufacturer’s protocol. Briefly, all reagents, working solution, standard solution and samples were prepared as instructed, 100 μl of standard or sample was added to each well, and the plates were incubated for 2 h at 37°C. Then, 100 μl of biotin-antibody (×1) was added to each well, incubated for 1 h at 37°C, aspirated from each well and washed, and 100 μl of HRP-avidin (×1) was added to each well. The plates were incubated for 1 h at 37°C, and 90 μl of TMB substrate was added to each well and kept for 15 min at 37°C protected from light. Finally, each well was filled with 50 μl of stop solution, the plate was gently shaken to ensure thorough mixing, and a microplate reader (BioTek; United States; IL) was applied to measure the absorbance at 450 nm.