BrdU Proliferation Assay

Primary lung-resident fibroblast proliferation was assessed using a BrdU Cell Proliferation Assay Kit (Cell Signaling Technology, Denver, CO, United States) as described (Sontake et al., 2017, 2018). Briefly, primary lung-resident fibroblasts were treated with DMSO or nintedanib (0.1, 0.5, and 1 μM) for 24 h then incubated with BrdU labeling solution for another 24 h along with nintedanib or DMSO. The cells were fixed 24 h after BrdU labeling, and immunodetection of BrdU was performed according to the manufacturer’s protocol. Change in proliferation was calculated as fold difference over control by measuring absorbance at 450 nm.