Bml277

The following antibodies were used for Western blotting or immunoprecipitation: anti-SF3B1 (Bethyl Laboratories, A300-996A-T, MBL International, D221-3), rabbit anti-cleaved Notch1 (Cell Signaling Technology, 4147S), rabbit anti–β-actin (Cell Signaling Technology, 4967S), rabbit anti-USP7 (Bethyl Laboratories, A300-033A), rabbit anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Cell Signaling Technology, 2118S), rabbit anti-CHEK2 (Bethyl Laboratories, A300-618A), mouse immunoglobulin G2b (IgG2b) (MBL International, M077-3), P5091 (Selleck Chemicals, S7132), PR619 (Sigma-Aldrich, SML0430), b-AP15 (Selleck Chemicals, S4920), ML323 (Sigma-Aldrich, SML1177), BML277 (MedChem Express, HY-13946), BML277 (Sigma-Aldrich, C3742, in vivo), SCH900776 (MedChem Express, HY-15532), camptothecin (Selleck Chemicals, S1288), flavopiridol (Sigma-Aldrich, F3055), topotecan (Sigma-Aldrich, T2705), etoposide (Sigma-Aldrich, E1383), mitoxantrone (European pharmacopoeia reference standard, M2305000), doxorubicin (Sigma-Aldrich, 44583), and E7107 and H3B-8800 (H3 Biomedicine).

All inhibitors were dissolved in dimethyl sulfoxide (catalog no. D8418, Sigma-Aldrich, Poole, UK) at a stock concentration of 10 mM for in vitro use. In preliminary experiments, the optimal concentration of CCT241533 (referred to as Chk2i from here; 10 μM; catalog no. CAY19178, Cambridge Bioscience, Cambridge, UK), BML-277 (5 μM; catalog no. 220486, Sigma-Aldrich, Poole, UK), and prexasertib (10 μM; catalog no. HY-18174A, Cambridge Bioscience, Cambridge, UK) that promoted DRGN/RGC survival and neurite outgrowth was determined. The Chk1 inhibitor LY2603618 (referred to from here as Chk1i; catalog no. 6454, Tocris, Oxford, UK) had no effect on DRGN/RGC survival at 1 to 50 μM, and hence, we used 20 μM, which was shown to induce DNA damage in a variety of human lung cancer cell lines including A549 and H1299 (53 (link)).

Cells were grown under different treatments as indicated in the figures. Colcemid Gibco, catalogue number 15212012; used at 0.27 µM. Topoisomerase I inhibitor: Camptothecin (CPT), C9911, Sigma; used at 2.5–10 µM. CDK1 inhibitor: RO3306, 4181, Tocris; used at 10 µM. CDC7 inhibitor: PHA‐767491, 3140, Tocris; used at 10 µM. ATM inhibitor: KU60019, CAY17502‐1, Cambridge bioscience; used at 10 µM. ATR inhibitor: AZ20 5198, Tocris; used at 10 µM. CHK2 inhibitor: BML‐277, Sigma; used at 10 µM. MRE‐11 inhibitor: mirin M9948, Sigma; used at 20 µM.