Azd7762

All animal experiments were performed with approval from the Peter MacCallum Cancer Centre Animal Experimentation Ethics Committee. C57Bl/6 mice (Walter and Eliza Hall Institute, Parkville, VIC, Australia) were intravenously injected with 2 × 10⁵ Eμ-Myc B-lymphoma cells in PBS and treated with pharmacological inhibitors from 8 days post-injection. Treatment of mice was continued until an ethical end-point was reached; hunched posture, ruffled fur, enlarged lymph nodes, laboured breathing, weight loss greater than 20% of start body weight and limited mobility or paralysis.
For use in vivo CX-5461 was prepared in 25 mM NaH₂PO₄ (pH4.5) and given by oral gavage at 30 mg/kg every three days. AZD7762 (Medchem Express) was delivered intraperitoneally in 10.3% -hydroxypropyl-β-cyclodextrin in 0.9% saline at 20 mg/kg daily on weekdays.
Reverse transcription qPCR, Western blot analysis, ChIP, Immunofluorescence-fluorescent in situ hybridisation (IF-FISH), psoralen crosslinking and chromatin accessibility by real time-PCR (CHART-PCR) assays were carried out as described previously [58 (link), 59 (link)]. A brief summary of these assays and lists of antibodies and primer sequences are provided in the Supplementary Data.

Poly-2-hydroxyethyl methacrylate (polyHEMA, P3932) and propidium iodide (PI, P4170) were purchased from Sigma-Aldrich. The ERK inhibitor SCH772984 was purchased from SelleckChem (S7101). CFSE dye was purchased from Tonbo Biosciences (10140–976 VWR). The Wee1 inhibitor MK-1775 (HY-10993) and CHK1 inhibitor AZD-7762 (HY-10992) were purchased from MedChem Express.

Primary CD4⁺ T cells were cultured in RPMI-1640 medium supplemented with 10% FBS, HEPES (10 mM), sodium pyruvate (1 mM), β-mercaptoethanol (50 μM), penicillin, and streptomycin. Naïve CD4⁺ T cells were isolated from spleens and lymph nodes of 8-week-old WT and Ddb1^fl/fl; OX40-cre mice, and cells were co-cultured with Antigen Presenting Cells (APC cells) in 96-well U bottom plate with soluble 1 μg/ml anti-mouse CD3 and 1 μg/ml anti-mouse CD28 antibodies. CD4⁺ T cells were activated for 0–6 days and harvested for flow cytometry analysis or western blotting.
Experiment to restore the phenotype of Ddb1 deficient activated CD4+ T cells and CD4⁺ T cells were treated with various inhibitors in CD4⁺ T-cell stimulation assay. Z-VAD (cat #HY-16658B), Z-IETD (cat #HY-101297), Ferrostatin-1 (cat # HY-100579), GSK872 (cat #HY-101872), Nec1 (cat # HY-15760), AZD7762 (cat # HY-10992), and Rabusertib (cat # HY-14720) were purchased from MedChemExpress; Nec-1s (cat #S8641) was purchased from Selleck.