Camptothecin cpt

Colorectal cancer (CRC) cell lines (HCT116, HT29, WIDR and T87), lung cancer cell lines (A549, H460, H1299 and H1975), a human bronchial epithelial cell line (BEAS-2B), human glioma cell lines (U251, U373 and U87), human hepatoma cell lines (HuH7, 7721, HepG2) and human embryonic kidney 293 cells (HEK 293T) were all purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The culture conditions used in this study are shown in Supplementary Table S1. High glucose Dulbecco’s modified Eagle’s medium (DMEM) and RPMI-1640 medium were supplied by HyClone (Thermo Fisher, USA). All culture media were supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher), 100 U/mL penicillin and 100 µg/mL phytomycin (Gibco, Thermo Fisher). All cultures were maintained at 37 °C and 5% CO₂. For induction of DNA damage, cells were treated with camptothecin (CPT, 1 µM, Selleck) for 1 h, etoposide (ETO, 100 μg/mL, Selleck) for 4 h, or γ-irradiation. ATM inhibitor (ATMi, KU-55933), ATR inhibitor (ATRi, VE-821) and DNA-PK inhibitor (DNA-PKi, NU7441) were purchased from Selleck. During DNA damage induction by ionizing radiation (IR), CPT or ETO, the corresponding inhibitors were maintained in complete medium continuously unless indicated.

Heme, radioimmunoprecipitation assay (RIPA) buffer and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Camptothecin (CPT) was obtained from Selleck Chemicals (Houston, TX), while protease and phosphatase inhibitor cocktail were purchased from Thermo Fisher Scientific (Rockford, IL). Mouse β III tubulin [also called TUJ1; Covance (Burlington, NC)] and anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from BioLegend (San Diego, CA). A rabbit sex determining region Y box (SOX)-2 monoclonal antibody was purchased from Millipore (Chemicon) (Temecula, CA), while forehead box g1 (FOXG1), NRG-1 and ERBB4 rabbit monoclonal antibodies were purchased from Abcam (Cambridge, MA). CXCL-10 was obtained from PeproTech (Rocky Hill, NJ), CXCR3 was obtained from MyBiosource (San Diego, CA) and BDNF was obtained from Thermo Fisher Scientific.

Cells were grown on coverslips and fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton-X-100 in PBS, blocked with 0.4% BSA, and incubated with appropriate primary antibody at 4 °C overnight. The slides were then washed with PBS containing 0.1% Triton-X-100 followed by incubation with fluorescently labeled secondary antibody at room temperature for 1 h. Meanwhile, 0.1 μg/ml DAPI (Sigma) was added to stain nuclei and 100 nM Mitotracker Red (Cell Signaling Technology) was used to stain mitochondrion. To analyze the level of activated caspase-3, transfected HepG2 cells were treated with 1 μM camptothecin (CPT, Selleck) for 48 h prior to staining with anti-cleaved caspase-3 (Cell Signaling Technology). The images were visualized using an Olympus or Zeiss confocal microscope. The relative fluorescence intensity was analyzed by Image J software. Each experiment was performed at least three times.