Phospho chk1

Salternamide A (SA, Figure 1A) was dissolved in 100% DMSO and stored at −20 °C for subsequent analysis. Cobalt (II) chloride (CoCl₂) and MG132 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for HIF-1α, Akt, phospho-Akt (Thr³⁰⁸), PI3K, phospho-PI3K (Tyr^458/199), RPS6, phospho-RPS6 (Ser^235/236), p70S6K1, phospho-p70S6K1 (Thr³⁸⁹), phospho-STAT3 (Tyr⁷⁰⁵), mTOR, phospho-mTOR (Ser²⁴⁴⁸), 4E-BP1, phospho-4E-BP1 (Thr^37/46), eIF4E, phospho-eIF4E (Ser²⁰⁹), phospho-CDC2 (Thr¹⁶¹), CDC25C, phospho-CDC25C (Ser²¹⁶), Chk1, phospho-Chk1 (Ser³⁴⁵), phospho-Chk2 (Thr¹⁶⁸), caspase-3, caspase-8, caspase-9, cleaved caspase-3, cleaved caspase-8, and LC3B were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies for ERK 1/2, phospho-ERK 1/2 (Thr²⁰²/Tyr²⁰⁴), STAT3, CDC2, cyclin B1, cyclin A, Bcl-2, and Bcl-xL were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for VHL, PARP, cleaved PARP, and Bim were purchased from BD Pharmingen™ (BD Biosciences, San Jose, CA, USA). Hsp90 antibody was purchased from Stressgen Bioreagents (Ann Arbor, MI, USA).

Protein extracts for immunoblotting were prepared by incubating cells in RIPA buffer (Boston BioProducts), supplemented with protease and phosphatase inhibitors (Halt Protease & Phosphatase Inhibitor Cocktail, EDTA-free; ThermoFisher Scientific), for 20 min. Supernatants were collected after centrifugation, 17,000 r.c.f. for 15 min, at 4^o C. The BCA reagent (Pierce) was used to determine the protein concentrations in the samples. SDS-PAGE was used to separate proteins, which were then transferred to polyvinylidene difluoride membranes (Millipore). Antibodies to the following proteins were used in the immunoblots: phospho-Histone H2A.X (Ser139, Cell Signaling, #9718, 1:1000), phospho-CHK1 (Ser345, Cell Signaling, #2348, 1:1000), CHK1 (Cell Signaling, #2360, 1:1000), SLFN11 (Santa Cruz Biotechnology, sc-374,339), and Actin (Cell Signaling, #4970, 1:1000).

Immunoblotting and subcellular fractionation were performed as previously described [12 (link)]. PFN1 (Abcam; ab118984, 1:2000), PTEN (Cell Signaling; 9188, 1:1000), Phospho-PTEN (Ser380/Thr382/383) (Cell Signaling; 9549, 1:1000), AKT (Cell Signaling; 4691, 1:1000), Phospho-Akt (Ser473) (Cell Signaling; 4060, 1:1000), α-Tubulin (Cell Signaling; 2144, 1:2000), SP1 (Santa cruz biotechnology; sc-59X, 1:2000), γH2AX (Abcam; ab11175, 1:2000), Chk1 (Santa cruz biotechnology, sc-377231, 1:1000), Phospho-Chk1 (Ser317) (Cell Signaling; 2344, 1:1000), Chk2 (Santa cruz biotechnology; sc-9064, 1:1000), Phospho-Chk2 (Thr68) (Novus Biologicals; NB100-92502, 1:1000), PARP (Cell Signaling; 9532, 1:1000), Cleaved-PARP(Asp214)(D64E10) (Cell Signaling; 5625, 1:1000) and β-actin (Abcam; ab54724, 1:2000), respectively followed by incubation with IgG-HRP (1:3000, Abcam, UK). Protein bands were visualized by Lumi femto solution (Dogen, Korea). Image J (National Institutes of Health, Bethesda, MD, USA) was used to quantify bands and compare to the loading control.

Cells were treated with DMSO, TMZ (100 μM), ABT-888 (100 μM), or the combination of TMZ (100 μM) and ABT-888 (100 μM) and harvested at various time points. Whole cells were lysed in standard NP-40 lysis buffer (Life Technologies) with 1X protease inhibitors tablet, 0.1 mM NaVO₃, 1 mM DTT, and 1 μM PMSF (Roche Applied Science). Samples were subjected to SDS-PAGE gel electrophoresis using standard protocols transferring 30 μg of protein onto a PVDF membrane (Millipore), followed by blocking with 10% nonfat milk powder for 30 min and overnight incubation at 4°C with either anti-MSH6 (Cat# 610918, BD Biosciences), anti-actin (Cat# MAB1501R Millipore), phospho-CHK1 (Ser 317, Cat# 12302S, Cell Signaling Technologies), CHK1 (Cat# 2360S, Cell Signaling Technologies) phospho-histone H2A.X (Ser 139, 80312S, Cell Signaling Technologies), or H2A.X (Cat# 2595S, Cell Signaling Technology) occording to manufacturer’s specifications. Membranes were incubated with appropriate secondary antibody for 1 h followed by enhanced chemiluminescence visualization and substrate detection (Thermo Scientific).