Anti cdc25c

Human NSCLC cell line A549 and H460 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were routinely cultured in RPMI 1640 medium (Gibco, Eggenstein, Germany) containing 10% heat-inactivated fetalbovine serum (Gibco, Eggenstein, Germany), 100 units/mL penicillin, and 100 ug/mL streptomycin in a humidified cell incubator with an atmosphere of 5% CO₂ at 37 °C. Antibodies including anti-MDM2 (sc-965, 1:200), anti-Cdc2 (sc-54, 1:200), anti-Cdc25C (sc-13138, 1:200) anti-Cyclin B1 (sc-245, 1:200), anti-Bcl-2 (sc-7382, 1:200), anti-Bcl-xL (sc-7382, 1:200), anti-cleaved PARP-1 (sc-56196, 1:200), anti-GAPDH (sc-293335, 1:1000), goat anti-mouse IgG-HRP and donkey anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies including anti-ATF4 (#11815, 1:1000), anti-p-EIF2α (#3398, 1:1000) and anti-EIF2α (#9722, 1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA).
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), Licochalcone A and DMSO (dimethyl sulfoxide) were purchased from Sigma (St. Louis, MO, USA); FITC Annexin V apoptosis Detection Kit I and PI (propidium iodide) were purchased from BD Pharmingen (Franklin Lakes, NJ, USA).

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).

The expression of proteins in cells were determined using western blot analysis as previously reported [15 ]. In this study, the primary antibodies were anti-14-3-3 σ (Santa Cruz, Dallas, TX, USA), anti-β-actin (Santa Cruz), anti-ATM (Santa Cruz), anti-ATR (Santa Cruz), anti-Cdc2 (Abcam, Cambridge, MA, USA), anti-Cdc25c (Santa Cruz), anti-Chk1 (Santa Cruz), anti-Chk2 (Santa Cruz), anti-cyclin B1 (Abcam), anti-p53 (Cell Signaling Technology Inc, Danvers, MA, USA), anti-phospho-p53 Ser 15 (Cell Signaling Technology Inc), anti-phospho-ATM (Santa Cruz), anti-phospho-ATR (Santa Cruz), anti-phospho-Cdc2 (Santa Cruz), anti-phospho-Chk1 (Santa Cruz), anti-phospho-Chk2 (Santa Cruz), anti-β-tubulin (Abcam), and anti-Wee1 (Santa Cruz) antibodies. An enhanced chemiluminescence (ECL, Sigma-Aldrich) system was used for developing signals of the blots, which were analyzed using a LAS3000 system (Fujifilm, Tokyo, Japan).

Lysate harvesting and western blot analysis was performed as previously described [57 (link)]. To quantify the levels of keratin 10 (K10), insoluble cell pellets obtained from RIPA-SDS lysis were resuspended in 8 M urea, 10% β-mercaptoethanol, 2 mM PMSF, and incubated at room temperature while rotating for 30 min, as described previously [90 (link)]. The insoluble debris was removed by centrifugation at 14,000 rpm at 4’C. Primary antibodies used: anti-SETD2 (Kind gift from Dr. Brian Strahl, Epicypher), anti-H3K36me3, anti-cyclin B, anti-CDK2 (Abcam), anti-H3.1 (Active Motif), p84 (GeneTex), anti-Involucrin, anti-keratin 10, anti-CDC25C, anti-cyclin A, anti-cyclin E and anti-GAPDH (Santa Cruz), anti-CDK1, anti-RPA32 (Bethyl laboratories) and anti-Flag (Sigma). Secondary antibodies used were horseradish peroxidase (HRP)-conjugated anti-rabbit (Cell Signaling Technology) and HRP-conjugated anti-mouse (GE Life Sciences). Western blots were developed using Clarity Western ECL blotting substrate (Bio-Rad). Images were captured on either autoradiography film or Biorad ChemidocMP imaging system. Blots were analyzed with Biorad Imagelab 5.0 software.