NU 7441

Proteasome inhibitor MG132 (HY-13259), Nutlin-3 (HY-50696), Cytarabine (HY-13605), BMS-536924 (HY-10262), AZ628 (HY-11004), and palmitic acid (HY-N0830) were purchased from MedChemExpress. Cycloheximide (R750107), Hydroxylamine (HAM, 467804), 2-BP (238422), and DAPI (D9542) were purchased from Sigma-Aldrich. iCRT14 (sc-362746) was purchased from Santa Cruz. A dual-luciferase reporter assay kit (DL-101-01) was purchased from Vazyme. N-Ethylmaleimide (NEM, A600450-0005) and BMCC-biotin ((1-Biotinamido)-4-[4′-(maleimidomethyl)cyclohexanecarboxamido]hexane, C100222-0050) were purchased from Sangon Biotech. Human palmitic acid ELISA kits (MM-51627H2) were purchased from MeiMian (Jiangsu, China). Anti-Flag agarose beads (23101) and Nu-7441 (503468-95-9) were purchased from Selleck (Houston, USA). RNase A (CW2105) was purchased from CWBIO. All antibodies used in this study are indicated in Supplementary Table S4. The human DUSP14, ACOX1, CTNNB1, and c-Myc coding sequences were amplified from HEK293T cDNA and cloned into pCMV-HA and pHAGE-CMV-MCS-PGK vectors. The human GSK3β and CK1 coding sequences were amplified from HEK293T cDNA and cloned into the pHAGE-CMV-MCS-PGK vector. The human ACOX1-TBE and DUSP14-RE were amplified from HCT15 gDNA and cloned into the pGL3-basic luciferase vector. The mouse ACOX1 coding sequence was amplified from mouse colon cDNA and cloned into pCDH-CMV-MCS-EF1-GFP+Puro vector. Mutations in the DUSP14, ACOX1, β-catenin, and Ubiquitin cDNAs were generated by overlap extension PCR. Deletion mutants from DUSP14 and ACOX1 were cloned into the pHAGE-CMV-MCS-PGK vector. Human DUSP14, CTNNB1, ACOX1, and mouse Acox1 shRNAs were designed and synthesized by RuiBiotech (Guangzhou, China), subsequently annealed, and inserted into the pLKO.1-puro vector. All primers for construction are presented in Supplementary Table S5.

Cell lines were treated with the following agents, either alone or in combination as indicated in text. DNA-PK inhibitors: NU7441 (Selleckchem), M3814 (Merck); KIT signaling inhibitors: dasatinib (Cayman Chemical), ibrutinib (Selleckchem), FTY720 (Cayman Chemical), and AAL(S) (synthesized by A/Prof Jonathan Morris, School of Chemistry, UNSW as described (43 (link))). Dimethyl sulfoxide was used as the solvent for all compounds. Final vehicle concentration was below 0.1% for all experiments.

(i) DNA damage inducer and PI3KK inhibitors. Hydroxyurea (HU; #127-07-1; MilliporeSigma) was prepared as a stock solution at 200 mM, according to manufacturer’s instructions. PI3KK inhibitors, KU60019 (#4176; Tocris Bioscience), AZ20 (#S7050; Selleckchem), and NU7441 (#3712; Tocris Bioscience), were dissolved in DMSO at 10 mM. They were applied to cell cultures at 2 days prior to infection at the final concentrations, as we previously reported: HU at 5 mM, KU60019 at 5 μM, AZ20 at 3 μM, and NU7441 at 1 μM (31 (link), 32 (link), 80 (link)).
(ii) In vitro DNA replication inhibitors. We used HAMNO (#S0148, Selleckchem), T2AA (#21921, Cayman Chemical), aphidicolin (#14007, Cayman Chemical), MK886 (#1311, Tocris Bioscience), and PNR7-02 (#2965, Axon Medchem). Stock solutions were prepared in DMSO at 10 mM. The inhibitors were directly added into the reaction of in vitro replication assays at a final concentration of 100 μM for HAMNO, 1 μM for T2AA, 10 μg/μL for aphidicolin, 200 μM for MK886, and 20 μM for PNR7-02 as previously recommended (44 (link)– (link)48 (link)).