2.7 MTT Experiment, Apoptosis Assay, Reactive Oxygen Species (ROS), and Mitochondrial Membrane Potential (ΔΨm) Detections

Cells were seeded in 96-well plates with appropriate number. Overnight, various doses (0–100 μl/ml) of BBJ and 200 μl/ml normal saline (NS) or nintedanib (NTB) were added. After 24, 48, and 72 h, cells were incubated with MTT for 3 h, the supernatant was discarded, and 150 μl/well DMSO was added. The OD value was detected at the length of 570 nm.

Cells were seeded in six-well plates. After adhering to the plates, cells were treated with BBJ and/or TGF-β1 for 24 h. For apoptosis assay, apoptotic cells were tested by an apoptosis kit (KeyGen Biotech, Nanjing, China) and analyzed by a NovoCyte™ flow cytometer (ACEA Bioscience, Inc., CA, USA). For intracellular ROS and ΔΨm detections, DCFH-DA and Rh123 were applied as related dyes and determined using flow cytometry.