Azd1775

Human ESCs were purchased from the American Type Culture Collection (ATCC) Company (Manassas, VA, USA), No. BFN608006424. Mouse endometrial stromal cells were prepared by zymogen digestion and differential adhesion. In short, mouse tissues were digested by zymogen and the target cells were obtained by different centrifugation speeds. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco) and 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma, St. Louis, MO, USA) at 37 °C in a humidified atmosphere of 5% CO₂.
The WEE1 inhibitor, AZD1775, was purchased from MCE (MedChemExpress; Monmouth, NJ, USA) and was used at a concentration of 40 mg/kg/day in in vivo studies. The β-catenin inhibitor, XAV939, was obtained from MCE and used at a concentration of 10 μM in in vitro studies. IL-1β was obtained from Sigma, and was used at concentrations of 5 ng/ml and 10 ng/ mL in cultured ESCs.

AZD1775 (adavosertib), birinapant (TL32711), and ASTX660 (tolinapant) were purchased from MedChemExpress at a stock concentration of 10 mM dissolved in dimethyl sulfoxide (DMSO). Tumor necrosis factor α with carrier (TNFα) was purchased from Biotechne R&D Systems and dissolved in 1% Bovine Serum Albumin in PBS at a stock concentration of 40 μg/mL.

AZD-1775, PF-00477736 and methotrexate were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Clofarabine was obtained from Sigma-Aldrich (Darmstadt, Germany). Both NALM-6 and RPMI-8402 were cultured in complete RPMI-1640 (Invitrogen, Waltham, MA, USA) medium containing 1% l-glutamine (Sigma-Aldrich), penicillin (100 U/mL, Gibco, Waltham, MA, USA) and streptomycin (100 μg/mL, Gibco) and 10% fetal bovine serum (Gibco).

AZD-1775 was purchased from MedChemexpress. Clofarabine, doxorubicin, imatinib, and ponatinib were obtained from Sigma-Aldrich. Bosutinib (Bos) was purchased from Tocris, and Bosutinib isomer (Bos-I) was purchased from LC Labs. Human B and T cell precursor ALL (B/T-ALL) cell lines (B-ALL: BV-173, SUP-B15, REH, NALM-6, NALM-19; T-ALL: MOLT-4, RPMI-8402, CCRF-CEM) were cultured in RPMI-1640 (Invitrogen) with 1% l-glutamine (Sigma-Aldrich), penicillin (100 U/ml, Gibco), and streptomycin (100 μg/ml, Gibco) supplemented with 10–20% fetal bovine serum (FBS, Gibco). All the cell lines were purchased from Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ) website (https://www.dsmz.de).

U2OS cells were grown in DMEM media (Gibco Cat #11965-092) +10% FBS (EqualFetal, Atlas Biologicals). Cell lines were cultured at 37°C, 5% CO2. Adovasterib (aka AZD1775; MedChemExpress) was resuspended in DMSO. Cells were treated with adovasterib as indicated.