Research Paper Volume 16, Issue 13 pp 10694—10723

Figure 3. Bleomycin-damaged primary alveolar epithelial cells exhibit cellular senescence, SASP, and an aberrant phenotype. (A) Following one-week culture post-bleomycin challenge, primary AECs were either fixed for high content immunocytochemistry analysis of nuclear p21 intensity and γH2A.X foci per nucleus or lysed for qPCR gene expression analysis of senescence genes Cdkn1a and Pai1. For gene expression, data were normalized to Gapdh housekeeper expression using the 2^−ΔCt method. (B) Supernatants from AECs were assayed for components of the SASP (IL-6, IL8, and IL-1β) by MSD kits and EGF by Luminex. (C) EdU incorporation was assessed by high content fluorescence analysis in bleomycin-treated and -untreated AECs to show arrest of the cell cycle by determination of Alexa647-positive cells. (D) SAβG activity was measured using a fluorogenic probe incubated on fixed cells at pH 6 for and subsequent nuclear staining with Hoechst, high content imaging, and quantitation in Harmony to calculate the percent positive objects. (E) AECs were lysed for qPCR gene expression analysis of common epithelial, mesenchymal, or aberrant basaloid markers. Data were normalized to Gapdh expression, a fold change value versus untreated AECs was determined using the 2^−ΔΔCt method, and fold change data were plotted in a heatmap. (F) Representative 10X magnification brightfield images of AECs with and without 24-hr bleomycin treatment at days 3 and 6. Data are representative of three biological donors across 3-5 independent experimental repeats. For all experiments, statistical analysis was performed using unpaired t-tests in GraphPad Prism: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001. Error bars represent SEM of n = 3 (A, E), n = 3-5 (B), or n = 2 (C, D, F) technical replicates.