Ape1 sirna

CDDP was purchased from Selleckchem Inc. (Houston, TX, USA). ¹³C₆-L-lysine, L-lysine, ¹³C₆¹⁵N₄-L-arginine, L-arginine, Dulbecco’s modified Eagle’s medium (DMEM)/F12 for SILAC, APE1 siRNA, dimethyl sulfoxide (DMSO), 2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin, and Dulbecco’s phosphate-buffered saline (PBS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 6-Diamidino-2-phenylindole (DAPI), Opti-minimal Essential Medium (MEM), Lipofectamine 2000, and the negative control siRNA were purchased from Invitrogen Inc. (Carlsbad, CA, USA). The Annexin V-phycoerythrin (PE) apoptosis detection kit was purchased from BD Biosciences Inc. (San Jose, CA, USA). The Cyto-ID® Autophagy detection kit was obtained from Enzo Life Sciences Inc. (Farmingdale, NY, USA). The Western blotting substrate, Pierce™ bicinchoninic acid (BCA) protein assay kit, skim milk, and radioimmunoprecipitation assay buffer (RIPA) were purchased from Thermo Fisher Scientific Inc. (Hudson, NH, USA). The polyvinylidene difluoride (PVDF) membrane was obtained from Bio-Rad Inc. (Hercules, CA, USA). The antibody against human β-actin was obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The remaining primary antibodies for signalling proteins related to apoptosis and autophagy were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).

Log phase HEK293 cells were transfected with NEIL1 siRNA (80 nM, targeting the 3′ UTR region of the NEIL1 gene; sense sequence, 5′-CCGUGAUGAUGUUUGUUUAUU-3′; antisense sequence, 5′-UAAACAAACAUCAUCACGGUU-3′; Sigma, St. Louis, MO), SAF-A siRNA (Dharmacon, catalogue number J-013501-05), APE1 siRNA (80nM, cat#SASI_Hs01_00027147, Sigma), individually or together for 48 h. Scrambled siRNA was used as a control. Downregulation of target genes was confirmed by immunoblotting of the cell extracts 48 h after transfection. Cells were irradiated with 2 Gy, 4 Gy, and 6 Gy x-rays and transferred to 6-well plates (300 cells/well) in triplicate. After allowing the cells to grow in fresh medium for 7-10 days, the colonies were stained with 0.5% crystal violet and counted.