Normal hbecs

Normal hBECs, from a male donor in their 60’s, were purchased from Lonza (Portsmouth, NH, USA) were grown in basal bronchial epithelial cell growth medium (BEGM) with the BEGM bullet kit (Lonza). Primary hLFs were isolated as described [11 (link)], (from excess de-identified control patient lung tissue of a female in their 70’s, whose use was approved by the University of Minnesota Institutional Review Board IRB#0611E97107), grown in DMEM, 10% FBS, and 1% P/S, and used at Passage 2. The cells were grown to confluence, trypsinized, and counted for inoculation into decellularized lungs. Human cells were utilized so that inoculated cell contributions to matrix remodeling could be distinguished from the pre-existing mouse matrix composition.

Normal HBECs (Lonza, Basel, Switzerland) were expanded to passage 4 in PneumaCult Ex Plus medium (STEMCELL Technologies, Vancouver, BC, Canada) and transferred to 24-well plates with transwell inserts for differentiation at the air–liquid interface (ALI) using PneumaCult ALI medium (STEMCELL Technologies, Vancouver, BC, Canada). Cells were cultured at the ALI for 25 days at 37 °C and 5% CO₂ with media changes every 2 to 3 days. On day 25, cells were rinsed with DPBS before apically treating with 25 uL of AbundMix (75%, 50%, or 10%, n = 3, Table S2a) or ToxMix (25%, 10%, or 1%, n = 3, Table S2b) in DPBS with a 1% DMSO vehicle for 2, 6, 10, 24, or 48 h or apically treated with 25 µL of ToxMix, retene, BaF, BbF, BcF, triphenylene, BeP, or BghiP (10%, 5%, 1%, or 0.5%, n = 4) in DPBS with a 1% DMSO vehicle for 24 h (Table S2b). Apical wash and basal media were collected and stored at −80 °C. Tissues were collected from inserts in an RLT lysis buffer with 1% β-mercaptoethanol using the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany) and stored at −80 °C.

Human fetal lung fibroblasts (IMR-90) were obtained from the American Type Culture Collection (Manassas, VA, USA) and grown in minimum essential medium (MEM) (Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; MP Biomedicals, Santa Ana, CA, USA). Normal HBECs (Lonza, Basel, Switzerland) were grown in Airway Epithelial Cell Growth Medium with SupplementPack (PromoCell, Heidelberg, Germany).