Anti phospho chk2

For SDS‐PAGE/Western blot analysis, the protein lysates were prepared by lysis with Tris‐HCl buffer, NP‐40 (1%), PMSF (1 mmol/L), and inhibitor cocktail (Roche, Switzerland). Protein samples were separated by 4–20% SDS‐PAGE (BioRad, Berkeley, CA) and transferred onto PVDF membrane. Protein detection was performed with polyclonal antibodies: anti‐DNA PKC (1:200, abcam), anti‐ChK2 (1:1000, Cell Signaling), anti‐phospho ChK2 (1:1000, Cell Signaling), anti‐ATM (1:1000, abcam, Cambridge, UK), anti‐phospho ATM (1:1000, abcam), anti‐TS (1:200, abcam), anti‐ MITF (1:1000, abcam), anti‐TK1 (1:10000, abcam), anti‐Caspase3 (1:1000, Cell Signaling), anti‐cleaved Caspase3 (1:1000, Cell Signaling), anti‐PARP (1:1000, Cell Signaling), and anti‐cleaved PARP (1:1000, Cell Signaling). The secondary IgG coupled to HRP (1:2000, Cell Signaling) was visualized with enhanced chemiluminescence (ECL+, GE Healthcare, UK). Equal protein loading was controlled using GAPDH‐specific antibody (Ambion, Life Technologies, Carlsbad, CA) and secondary goat antimouse IgG linked to HRP (abcam). The experiments were carried out three times in triplicates. The bands were detected using the ImageQuant LAS 4010 camera system (GE Healthcare).

MTT was purchased from Amresco (Solon, OH, USA). The primary antibodies used in this study included the following; anti-PARP1, anti-cleaved caspase 3, anti-phospho-CHK1, anti-CHK1, anti-phospho-CHK2, anti-CHK2, anti-phospo-p53, anti-p53 (Cell Signaling Technology; Danvers, MA, USA), anti-Cyclin B1, anti-CDC25C, anti-phospho-histone H2AX (Santa Cruz Biotechnology; Dakkas, TX, USA), and anti-β-actin (Sigma-Aldrich; St. Louis, MO, USA) antibodies. Olaparib (AZD2281) and quizartinib (AC220) were purchased from Selleckchem (Houston, TX, USA) and dissolved in DMSO (Sigma Aldrich). N-acetyl-L-cysteine (NAC) was purchased from Sigma Aldrich. CM-H₂DCFDA was purchased from Invitrogen (Carlsbad, CA, USA).

Asyncronized DAOY and ONS-76 cells were treated with 4-HPR (5–10 μM) or Vincristine (50 μM) as positive control, for 24h trypsinized, and fixed in cold 70% ethanol. DNA was stained with 100 μg/ml propidium iodide (PI) (Sigma- Aldrich) in hypotonic citrate buffer with 20 μg/ml ribonuclease A. Stained nuclei were analyzed for DNA-PI fluorescence using a FACSCanto flow cytometer. Resulting DNA distributions G0/G1, S, G2/M and apoptotic phase of the cell cycle were analyzed with FACSDiva software. 4-HPR effects on cell cycle were also investigated by detection of phosphorylated ciclin-kinase2 (anti-phospho-Chk2, Cell Signaling Technology, Danvers, MA) levels by cytofluorimetric analysis. Cells treated with 10 μM Etoposide were used as positive control. Briefly, cells were detached, collected by centrifugation and fixed/permeabilized using Cytofix-Cytoperm solution (BD) for 10 minutes at 4°C at dark. Unconjungated phospho-Chk2 primary antibody was added to each tube for 1h at room temperature. Cells were then washed and incubated with the secondary antibody conjugated with Alexa Fluor 482 (Life Technologies, Monza, Italy) for 30 minutes at room temperature. Cells were then rinsed, resuspended in PBS 1X and analyzed by flow cytometry. Expression levels of Cyclin D1 and CDK4 were detected by Western Blot analysis (see below).

Mouse anti-Chk1, rabbit anti-Chk2, anti-H₂AX, anti-phospho-Chk1, anti-phospho-Chk2, and anti γ-H₂AX antibodies were purchased from Cell Signaling Technology (Danvers, USA). Mouse monoclonal anti-β-actin and anti-FLAG were purchased from Sigma (St. Louis, USA), and rabbit anti-TIPE2 was purchased from Boster (Wuhan, China). Puromycin, crystal violet, MTT, DMSO, and bovine serum albumin (BSA) were purchased from Sigma, and DMEM medium and fetal bovine serums were purchased from Gibco (Gran Island, USA). Lentiviral vector pLKO.1, psPAX2, and pMD2.G were obtained from Addgene.

Cells or tissue samples were lysed with RIPA lysis buffer (Beyotime Biotechnology) supplemented with phosphatase (PhosSTOP, Roche) and protease (cOmlpete, Roche) inhibitor cocktails. Immunoblotting was performed as described previously [42 (link)]. The following antibodies were used: anti-PLK1 (Cell Signaling Technology, Cat# 4513, RRID: AB_2167409); anti-Cleaved Caspase-3 (Cell Signaling Technology, Cat# 9661, RRID: AB_2341188); anti-phospho-Histone H2A.X (Millipore, Cat# 05-636, RRID: AB_309864); anti-phospho-CHK2 (Cell Signaling Technology, Cat# 2197, RRID: AB_2080501); anti-CHK2 (Cell Signaling Technology, Cat# 6334, RRID: AB_11178526); anti-GAPDH (Zhongshanjinqiao Biotechnology, Cat# TA-08, RRID: AB_2747414) and anti-beta-actin (Cell Signaling Technology, Cat# 4970, RRID: AB_2223172).