Rl95 2

Detailed methodology on isolation of HESCs can be found in supplemental Methods. HESCs were seeded in 96-well plates (2 × 10³ cells per well) and incubated in DMEM/F-12 containing 10% FBS and 24 μg/ml ABs. Ishikawa cells were purchased from Procell Ltd. (RL95-2, Wuhan, China) and cultured in DMEM/F12 plus 10% FBS in 96-well plates (1 × 10³ cells per well). The proliferative behaviors of HESCs and Ishikawa cells were evaluated using a cell counting kit-8 (CCK-8) assay (Dojindo, Shanghai, China) according to the manufacturer's protocol. Cells cultured by DMEM/F-12 with 10% FBS served as controls.
To evaluate the effect of ABs on migration of HESCs, cells were seeded into 6-well plates (3 × 10⁵ cells per well) and cultured in DMEM/F-12 containing 10% FBS until confluent monolayers. A linear wound was made using a 200 μL pipette tip. After washed with PBS, cells were incubated in DMEM/F-12 containing 10% FBS and 24 μg/ml ABs. The area of the scratch was measured at 0 h, 6 h and 24 h through taking six representative images. The closure rate was calculated as follows:
To evaluate the effect of ABs on fibrotic gene expression of HESCs, qRT-PCR on fibrotic genes (Col IA2, Fibronectin [FN], connective tissue growth factor [CTGF], and α-SMA) was performed, all primers are listed in Table S1, and data were normalized using β-actin expression (see supplemental Methods for details).

EC cell lines, including HEC-1-B, Ishikawa and RL95-2, and human endometrial stromal cells (hESC) were purchased from Procell (Wuhan, China) and cultured in 90% DMEM with 10% FBS, in a 37°C environment containing 5% CO₂.