Tvb 2640

BALB/c nude mice (4–6 weeks old, female, 14–16 g) were purchased from Beijing HFK Bioscience Co. Ltd. and housed under specific pathogen-free and controlled conditions (25–27, 45–55% humidity, 12 h day/night cycle). The study was approved by the Institutional Animal Care and Use Committee (IACUC) of the National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, and the methods were carried out in accordance with the approved guidelines.
The cells were subcutaneously injected into the flanks of mice to establish a xenograft tumor. When the size of tumors reached approximately 100 mm³, animals were randomly divided into four groups and intraperitoneally injected with 10 mg/kg etoposide (S1225, Selleckchem), or 8 mg/kg TVB-2640 (#S9714, Selleckchem), or etoposide (10 mg/kg) and TVB-2640 (8 mg/kg) combination, or 0.9% saline as control. Tumor length (a) and minor diameter (b) were monitored once a week, and tumor size was calculated using the following formula: volume=a×b²/2.

Human breast cancer cell line MDA-MB-231 was purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences). Cells were cultured in dulbecco’s modified eagle medium (DMEM) (KeyGen Biotech) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 100 U/mL antibiotics (penicillin and streptomycin) at 37 °C in a humidified incubator with 5% CO₂. When the cell confluence reached 80–90%, the TVB-2640 (Selleck, S9714) and/or PF-05175157 (Selleck, S6672) were added respectively. After 24 h, the cell viability was detected by CCK-8 assay and colony formation test, the cell proliferation was reflected with EdU Staining and DAPI nuclear staining, and the cell migration was monitored by wound healing assay.

Ovsaho cells were obtained from JCRB (Japanese Collection of Research Bioresources Cell Bank) cell bank (NIBIOHN, Cell No. JCRB 1046) and grown in MCDB105/199 medium with 10% FBS (Fisher, #10438026) and substituted with 1% Penicillin-Streptomycin. All cells were free of Mycoplasma and their identity was verified by whole exome sequencing at the Broad Institute. For dose-response curves, cells were treated for 72 h with DMSO or the indicated doses of the PARP1 inhibitor AG-14361 (Selleckchem, #S2178), FAS/FASN inhibitor TVB-2640 (Selleckchem, #S9714), Bortezomib (Selleckchem, #S1013), proTAME (Selleckchem, #S9605), GC7 (Sigma Aldrich #259545). In combination assays, inhibitor concentrations were combined in a 1:1 ratio for indicated total concentration. Cell viability was measured using the software in a live-imaging IncuCyte station by counting live cells. Live cells were labeled by adding the Incucyte NucLight Rapid Red (NRR) Reagent (Essen Bioscience, #4717, 1:4000) to the medium. Datapoints of three biological experiments and three technical replicates (total n = 9) were merged and fitted using a non-linear log(inhibitor) versus normalized response with variable slope for dose-response curves using Prism (GraphPad). Combination Index (CI) was calculated based on Chou-Talaly method [53 (link)].