Research Paper Volume 16, Issue 13 pp 10694—10723

Figure 4. Secreted factors from senescent alveolar epithelial cells potentiate pro-fibrotic gene expression and protein secretion more robustly than direct damage of fibroblasts. (A) Schematic summarizing the experimental protocol of direct damage of NHLFs with bleomycin or indirect treatment of NHLFs by transferring conditioned medium from bleomycin-treated AECs. Image created with https://www.biorender.com/. (B) Treated or untreated NHLF cells were lysed, and qPCR gene expression analysis was performed. Expression of senescence and fibrosis-related matrix and secreted factor genes was assessed, and data were normalized to β2m housekeeper expression using the 2^−ΔΔCt method. (C) Senescent or non-senescent AEC conditioned medium or blank AEC medium was diluted 1:3 in fibroblast medium and incubated on NHLFs for 5 days. Cells were lysed for gene expression analysis of senescence and fibrosis-related genes. (D) Supernatants were collected from the treatments described in C and assayed for fibrosis-related secreted proteins by MSD kits. Data are representative of three independent experiments accounting for 3 biological donors of AECs and 2 donors of NHLFs. Statistical analysis was performed using a two-way ANOVA and a Dunnett’s Multiple Comparisons post-test vs. control conditions in GraphPad Prism: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001, ns not significant. Error bars represent SEM of n = 2 technical replicates.