Cell culture and treatment

BEND bovine endometrial epithelial cells (bEECs; ATCC^® CRL-2398^™) were acquired from American Type Culture Collection. The cells were grown in a 1:1 mixture of Ham's F12 and Eagle's Minimal Essential medium (Gibco; Thermo Fisher Scientific, Inc.) with Earle's balanced salts with 1.5 mM L-glutamine (MilliporeSigma) adjusted to contain 1.5 g/l sodium bicarbonate (MilliporeSigma) supplemented with 0.034 g/l D-valine (MilliporeSigma), 10% FBS (Biowest LLC) and 10% horse serum (Gibco; Thermo Fisher Scientific, Inc.) in an environment containing 5% CO₂ at 37˚C. HKL (purity, ≥98%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., where its structural formula is shown in Fig. 1A.

Honokiol treatment improves the viability of LPS-treated bEECs. (A) Chemical structural formula of HKL. (B) The viability of bEECs following treatment with different doses of HKL as detected by MTT. (C) The viability of LPS-stimulated bEECs following treatment with different doses of HKL as detected by MTT. ^**P<0.01 and ^***P<0.001 vs. Control; ^##P<0.01 vs. LPS. HKL or Hon, honokiol; LPS, lipopolysaccharide; bEECs, bovine endometrial epithelial cells.

Pre-treatment of the cells was performed with HKL at doses of 1, 10 and 20 µM for 1 h at 37˚C, followed by stimulation with 1 µg/ml LPS for 12 h at 37˚C. In addition, 1 µg/ml Tunicamycin (an ER stress inducer; purchased from MedChemExpress), was added into cells for 2 h at 37˚C followed by LPS treatment. Cells without any treatment were considered to be the control group.