Penicillin streptomycin antibiotics

The HTR-8/Svneo cell line, derived from human chorionic cells [27 (link)], was obtained from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. The HTR-8/Svneo cells were cultured in Dulbeccós modified eagle medium (DMEM, Gibco, D5796, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, 10099141) and penicillin/streptomycin antibiotics (Beyotime Biotechnology, SV30010, China) and incubated in a 5% CO₂ incubator (Shanghai SANTN Co., Ltd., DH-160I, China) at 37°C.
To investigate the impact of TLR9 on trophoblast cells, HTR-8/Svneo cells were treated with either the TLR9 activator ODN1668 [28 (link)] or the TLR9 inhibitor HCQ. Then, the HTR-8/Svneo cells were exposed to 5 μmol/l ODN1668 or 10 μmol/l HCQ, respectively, while rats in the Control group were given an equivalent dose of media.

Human lung cancer cell lines PC-9 was obtained from the Institute of Cancer Prevention and Treatment, Harbin Medical University, and maintained in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin antibiotics (Beyotime). Cultures were incubated in a humidified atmosphere of 95% air and 5% carbon dioxide (CO₂) at 37 °C.

Normal human gastric epithelial cell line GES-1 and a panel of human gastric cancer cell lines, including AGS, HGC-27, KATO3, MKN45, N87, and SNU-1 were supplied by the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and grown in RPMI-1640 medium (HyClone, Logan, UT, USA) containing 10% fetal bovine serum (FBS) (Gibco, Gaithersburg, MD, USA) and 100 U/ml penicillin-streptomycin antibiotics (Beyotime, Beijing, China) in a humidified atmosphere containing 5% CO₂/95% air at 37 °C. Transgenic GC cell lines, including MKN45-NDUFS1 (MKN45 overexpressing NDUFS1), MKN45-mock (control for MKN45-NDUFS1), N87-shNDUFS1 (N87 interfering NDUFS1), N87-shcontrol (control for N87-shNDUFS1), MKN45-NDUFS1-FBLN5 (MKN45 overexpressing both NDUFS1 and FBLN5), MKN45-NDUFS1-NC (control for MKN45-NDUFS1-FBLN5), N87-shNDUFS1-shFBLN5 (N87 interfering both NDUFS1 and FBLN5) and N87-shNDUFS1-shNC (control for N87-shNDUFS1-shFBLN5) were generated as previously described [15 (link), 16 (link)].

Six 4-week-old S-D rats (3 males and 3 females; Zhuhai Bestest Biotechnology Co. Ltd., China) were euthanized by the inhalation of an overdose of 5% isoflurane. Briefly, cartilage was isolated from the knee joint of rats under aseptic conditions. Cartilage samples were cut into pieces, digested in 0.25% trypsin (C0201; Beyotime, China) at 37°C for 30 min, and then digested in 0.2% type II collagenase (C222; Sigma, USA) for 1 h at 37°C. Thereafter, the cells were filtered through a 200 mesh screen and centrifuged at 500 ×g for 8 min. Then, the cells were resuspended in DMEM containing 10% fetal bovine serum (16140089, Gibco, USA) and 1% penicillin/streptomycin antibiotics (100 X, C022, Beyotime, China) and cultured in a 5% CO₂ cell incubator at 37°C. trypsin (0.25%) was added to the chondrocytes when the cell density reached 80%. After centrifugation at 200 × g for 5 min, the cells were passaged at a ratio of 1:3. The second-passage chondrocytes were subjected to the following tests.

The human triple-negative breast cancer cell lines MDA-MB-231 and BT-549 were from the cell bank of the Chinese Academy of Sciences (Shanghai). MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s medium plus 10% fetal bovine serum (FBS, GIBCO, Brazil) and 1% penicillin-streptomycin antibiotics (Beyotime, China). BT-549 cells were cultured in RPMI 1640 medium (GIBCO, Brazil) plus 10% FBS and 1% penicillin-streptomycin. All cells were cultured in a 37°C incubator containing 5% carbon dioxide.

Normal human mammary epithelial cells (MCF10A) and the accepted BC cell lines (MDA-MB-468, SKBR3, MCF7 and MDA-MB-231) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were allowed to grow in Dulbecco modified Eagle medium supplemented with 10% (v/v) fetal bovine serum (PAN-Biotech, Aidenbach, Germany), 1% penicillin–streptomycin antibiotics (Beyotime, Shanghai, China) in a humidified atmosphere containing 5% CO₂ at 37°C. DMEM medium was changed every third day.