Bms303141

BT549 and Hs578T were cultured in RPMI 1640 medium (Gibco-BRL, Australia) containing 10% fetal bovine serum (FBS) (Gibco-BRL, Australia) at 37 °C in humidified atmosphere containing 5% CO₂ with 1% O₂ or 21% O₂. ATM, PFKP, or CS stable knocked down cell lines were produced by lentiviral-mediated transduction using synthetic short hairpin RNA (shRNA) oligonucleotides (GenePharama, Shanghai, China) according to the manufacturer’s protocols. The sequences of small interfering RNA and shRNA used were listed in Supplementary Table 1.
The PFKP promoter containing HIF1A-binding sites (wild type (WT): –GTACGTGGAG–) and its mutant sites (Mut: –GTAgaaaGAG–) were cloned into pGL3 luciferase reporter vector (GenePharma China) to construct the WT or Mut PFKP reporter plasmid. The reagent cisplatin was a gift from the First Affiliated Hospital of Chongqing Medical University; sodium citrate (10 mM, 12 h) was purchased from Sigma-Aldrich (St. Louis, MO, USA); KU60019 (10 μM, 12 h), MG132(40 μM, 12 h), BMS303141 (20 μM, 12 h), and SCH772984 (2 μM, 12 h) were from Selleck (USA). All experiments were performed at least three times.

U2OS DRGFP cells were plated in a 6 cm dish at a concentration of 2.5×10⁵ cells per well and left to attach overnight. Cells were treated with an ACLY inhibitor (BMS303141, Selleckchem, Munich, Germany) or Rad51 inhibitor (B02, Calbiochem) for 6 h and then transfected with 10 µg pCBASceI plasmid (Addgene #26477) using Lipofectamine 3000 reagent for 24 h. Medium was refreshed after 24 h and cells were harvested by trypsinisation 48 h after transfection. The fraction GFP positive cells was determined by flow cytometry using a FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA).

Male C57BL/6 N mice (7–10 weeks, 20–25 g) were obtained from Charles River (Beijing, China) and randomly divided into experimental groups (n = 5 per group). No statistical methods were used for the animal sample size. The mice were injected intraperitoneally with lipopolysaccharide (LPS, L2630, E. coli 0111:B4, Sigma Aldrich, MA, USA) (5 mg/kg body weight [59 (link), 60 (link)]) in 0.9% saline (0.9% NaCl) to induce endotoxemia. In the intervention group, the mice were intraperitoneally injected with BMS-303141 (S0277, Selleck, Shanghai, China) (50 mg/kg body weight) 1 h before LPS injection. Vehicle control mice were intraperitoneally injected with 100 µL of saline. After 16 h of LPS challenge, the mice were anesthetized and the blood samples were collected. The organs were snap frozen or fixed with formalin for further examination. The frozen organs were stored at −80 °C. Investigators were blinded to the group allocation for the analysis. The protocols for animal experiments were approved by the Animal Ethics Committee of Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine (No. 092) and were in line with the International Guidelines for Care and Use of Laboratory Animals (National Academy of Sciences Health Publication No. 85–23, revised in 1996).