Proliferative Capacity of HPAECs

The in vitro proliferative capacity of HPAECs was analyzed using three independent assays. MTT proliferation assay was performed as described previously. Briefly, HPAECs were suspended in EGM-2–containing EBM-2 and plated on flat-bottomed 96-well plates at a density of 1 × 10⁵ cells per well in triplicate. After overnight seeding (16 hours), cells were challenged with TSP1 (2.2 nM), and proliferation was assessed using MTT (Sigma-Aldrich) assay. Absorbance at 570 nm was read using a plate reader (BioTek Synergy 4 Hybrid Multi-Mode Microplate Reader), and the proliferation index was calculated relative to the absorbance value of the vehicle-treated controls. Trypan blue exclusion assay was performed as documented previously (76 (link)). Briefly, TSP1 (2.2 nM) was added to cultures of HPAECs for indicated periods after an overnight seeding and a 4-hour synchronization. Subsequently, cells were trypsinized, 10 μl of trypan blue (4% solution) was added to an equal volume of cell suspension, and cell numbers were counted using a hemocytometer. Cell numbers were normalized and expressed as a fold change over time from the start of the experiment (time, 0 hour). BrdU assay was performed as per the manufacturer’s instructions.