Doxycycline hydrochloride

Human NSCLC cell lines (A549, H1299, H157 and H520), normal bronchial epithelial cell line (HBE), mouse NSCLC cell line (LLC, Lewis lung carcinoma cells) and HEK293T cells were obtained from the Cell Bank of the Chinese Academy of Sciences. All human NSCLC cell lines were cultured in RPMI 1640 medium, whereas other cells were cultured in DMEM. FBS (10%) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) were added to the media. All cells were cultured in a constant temperature incubator (5% CO₂, 37℃). Cell transfection was performed using Lipofectamine 8000 according to the instructions.
The following reagents were used for cell experiments: Recombinant BMP-2 protein (Sino Biological, 10426-HNAE) and doxycycline hydrochloride (dox; Sangon, A600889). All cells were freshly thawed before experiments and cells older than 8 weeks were not used in the study. All cell lines were frequently assayed for Mycoplasma using Mycoplasma Stain Assay Kit (Beyotime) to ensure they were free of Mycoplasma contamination. NSCLC tissues and matched adjacent nontumor tissues were collected from Xiangya Hospital, Central South University, with informed consent from the patients. This study was approved by Xiangya Hospital, Central South University.

HO8910 and HEK293T cell lines were purchased from China Type Culture Collection (Shanghai, China). A2780, HEY, SKOV3 cell lines were from Jian-Jun Wei’s lab. HEY, HO8910, and A2780 cells were cultured in DMEM medium (Gibco, Invitrogen). SKOV3 cells were cultured in McCoy’s 5A medium. All media contained 10% FBS (Gibco, Grand Island, NY, USA). Doxycycline Hydrochloride (Sangon Biotech, Shanghai, China) was added to cell culture medium at the final concentration of 100 ng/ml for 48–72 h when induced expression of tet-on shRNA vector is needed. All cells were cultured at 37 °C with 5% CO₂ in a humidified incubator.