Mk 1775

The Library of Pharmacologically-Active Compounds (LOPAC), 6α-methylprednisolone (6MP), dexamethasone (Dex), mifepristone, PD166285 (PD16), PD173952 (PD17), NU6027 and CGP7454A were purchased from Sigma-Aldrich; dasatinib, MK-1775 and CHIR-124 from SelleckChem. All compounds were diluted in DMSO to 10 mM stock solutions and kept at -20°C.

JNJ-10198409 (JNJ) and Picropodophylin (PPP) were purchased from Calbiochem, Merck Millipore (Nottingham, UK). Propidium iodide, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), PDGF-BB, IGF-1, nocodazole, chloroquine, AEBSF, Ucf-101 and salubrinal were purchased from Sigma-Aldrich (St. Louis, MO, USA), 7-Amino-Actinomycin D (7-AAD) was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Z-VAD (OMe)-FMK (ICn), Z-D(OMe)E(Ome)VD(OMe)-FMK (IC3), Ac-IETD-CHO (IC8) and necrostatin-1 were purchased from Calbiochem, Merck Millipore (Darmstadt, Germany). MK-1775 was purchased from Selleckchem (Houston, TX, USA).

All pancreatic cancer cells were purchased from ATCC. MIA PaCa2, PANC-1, Hs 766T, HPNE, and Capan-1 cells were cultured in DMEM (Gibco/Invitrogen, Carlsbad, CA); BxPC3, Capan-2, PL11 and SU.86.86 cells were cultured in RPMI 1640. All cells were supplemented with 10% FBS (Gibco/Invitrogen) expect PL11 (15% FBS and insulin), 1% L-glutamine (Gibco/Invitrogen), and 1% penicillin-streptomycin (Invitrogen) at 37 °C in 5% humidified CO₂ incubators.
For transient transfections, siRNAs against FANCD2, BRCA2 and control oligos (Dharmacon) were transfected using lipofectamine 2000 (Gibco/Invitrogen) as previously described18 (link). All cells were harvested 48 hours post-transfection.
All cells were treated with the IC₅₀ values of the DNA damaging agent MMC (mitomycin C; (Sigma, St. Louis, MO) and oxaliplatin (Sigma) as previously described18 (link) by adding directly into the culture medium. MK-1775 was purchased from Selleckchem, Houston, TX.

The inhibitors used in this study were employed at the following concentrations: Roscovitine at 25 μm (Cdk inhibitor; Selleck Chemicals, Houston, TX, USA), NU6140 at 10 μm (Cdk2 inhibitor; Calbiochem), RO‐3306 at 10 μm (Cdk1 inhibitor; Calbiochem, Darmstadt, Germany), MG‐132 at 10 μm (Inhibitor of the proteasome; Sigma‐Aldrich, Saint Louis, Missouri, USA), BI2536 at 100 nm (Plk1 inhibitor; Selleck Chemicals), MK‐1775 at 100 μm (Wee1 inhibitor; Selleck Chemicals), Nutlin‐3 at 13 μm (Mdm2 antagonist; Sigma‐Aldrich), SB202190 at 10 μm (p38 inhibitor; Selleck Chemicals), Cycloheximide at 10 μg mL⁻¹ (Inhibitor of protein translation; Sigma‐Aldrich). Etoposide (topoisomerase II inhibitor; Sigma‐Aldrich) was employed at 1 μm, which is sufficient to induce robust checkpoint arrest in cells at all cell cycle stages (Müllers et al., 2014). Figure S1D (Supporting information) shows the robustness of the checkpoint arrest as well as the efficiency of Cdk inhibitor treatment.
SMARTpool ON‐TARGET plus siRNAs targeting CDKN1A (p21), CDK1, or CDK2 were purchased from Dharmacon and employed at a concentration of 20 nm using HiPerFect (Qiagen, Hilden, Germany) transfection at 48 and 24 h before analysis of the phenotype.

Cisplatin was purchased from Sigma (St. Louis, MO, USA). Prexasertib was provided by Eli Lilly or purchased from MedChemExpress (Monmouth Junction, NJ, USA). The Prexasertib provided by Eli Lilly was used to establish acquired‐resistant cell lines, evaluate the characteristics of the resistant cell lines, do the inhibition experiments by Wee1 inhibitor and detect the downstream proteins. All other experiments were performed with Prexasertib purchased from MedChemExpress. Several experiments were performed using both drugs with superimposable results. AZD7762, PF477736, RO3306, K3861, THZ1, BIRB796 and MK1775 were purchased from Selleckchem (Houston, TX, USA).