Bi2536

Patient-derived tumor cells, PDX366, were maintained in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and cultured in a humidified (37 °C, 5% CO₂) incubator. This line is established from a poorly differentiated metastatic tumor with low stromal content and mutant for KRAS, P53, and SMAD4 but WT for P16 genes, and it was authenticated by the University of Virginia Biomedical Research Facility in 2010 as previously described^{16 (link)}. PDAC cancer MPANC-96 and BxPC3 cells (ATCC, Manassas, VA) were cultured in RPMI-1640 medium supplemented with 10% FBS and %1 penicillin/streptomycin in a humidified (37 °C, 5% CO₂) incubator. Trametinib (EuroAsia TransContinental, Mumbai, India), GSK923295 (ChemieTek, Indianapolis, IN, USA), Aurora A/B inhibitor (ZM447439, Selleckchem, cat. no. S1103), and PLK1 inhibitor (BI-2536, MedChemExpress, cat. no. HY-50698) were dissolved in sterile DMSO to make 5 mM stock solutions. Aliquots of the stock solutions were stored at −20 °C.

Patient-derived GSCs and human neural progenitor cells (hNPC) were cultured in human neural stem cell maintenance media (Millipore), 1% penicillin-streptomycin (PS), and supplemented with EGF and β fibroblast growth factor (20 ng/ml each). hNPC was induced from human embryonic stem cells H1 as described previously [71 (link)]. Noncancerous mouse astrocytes (C8-D1A) were cultured with Dulbecco’s modified Eagle’s medium/nutrient mixture F12 (DMEM/F12) with 10% fetal bovine serum (FBS) and 1% PS. Human embryonic kidney (HEK) 293T cells were cultured with DMEM-high glucose with 10% FBS and 1% PS. The following inhibitors were used in this study: 17-AAG (MedChemExpress), Z-VAD-FMK (MedChemExpress), KN-93 (MedChemExpress), Volasertib (Selleckchem), BI 2536 (MedChemExpress), KU-55933 (MedChemExpress), and Nocodazole (Sigma).

The compound library used for the chemical screen was provided by the IRIC HTS facility and was assembled from various commercial sources and in house syntheses (http://www.iric.ca/en/research/core-facilities/high-throughput-screening/?section=technologies). Thymoquinone was obtained from Sigma-Aldrich, Poloxin and BI2536 from MedChem Express, and PPG from ChromaDex. See Supplementary Table S2 for all vendor information concerning other compounds. All chemical structures were drawn using ChemBioDraw software.

NALM-6, HEK293T and hTERT RPE-1 cells were cultured at 37°C with 5% CO₂; in RPMI medium (Wisent) for NALM-6 and in DMEM medium (Wisent) for HEK293T and RPE-1 cells, all supplemented with 10% fetal bovine serum (FBS, HyClone), penicillin and streptomycin (Wisent). SMARTpool ON-TARGETplus human KIF18A siRNA, human SKA1 siRNA, human PRR14L siRNA and ON-TARGETplus Non-targeting Pool were purchased from Horizon Discovery. SiRNA transfections in RPE-1 cells were carried out with Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer’s procedures. BI2536, BI6727/Volasertib and GSK461364A were purchased from MedChemExpress and Reversine was purchased from Cayman Chemical Co.

METTL3 knockdown and overexpression vectors were transduced in DPSCs, wihle PLK1 inhibitor BI2536 (1 nM, MedChemExpress) [24 (link)] or DMSO as negative control were used to inhibit PLK1 expression in shMETTL3-DPSCs. DPSCs were washed with PBS and then fixed in 70% cold ethanol overnight. At least 50,000 cells were subjected to propidium iodide staining with a Cell Cycle and Apoptosis Analysis Kit (Beyotime), and the DNA content was measured by fluorescence-activated cell sorting (FACS) instrument (BD Biosciences).