Small interfering rnas sirnas

RPMI 1640, fetal bovine serum (FBS), TRIzol^® reagent, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), and the platinum SYBR Green qPCR SuperMix-UDG were purchased from Invitrogen (Carlsbad, CA, USA). M-MLV Reverse transcription system and GoTaq^® qPCR Master Mix were from Promega (Madison, WI, USA). BCA Protein Assay Kit was from KeyGEN Biotech (Nanjing, China). siTRAN transfection reagent was from OriGene (Beijing, China). Primers and small interfering RNAs (siRNAs) were synthesized respectively by Invitrogen (Shanghai, China) or GenePharma (Suzhou, China). Anti-XPA (sc-853), anti-GAPDH antibodies and HRP-conjugated secondary antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), ECL Western blotting detection reagents was from Amersham Biosciences (Piscataway, NJ, USA). Peroxidase Envision Kit was from Dako (Carpinteria, CA, USA). Cisplatin and all other reagents were of molecular biology grade and obtained from Sigma-Aldrich (Shanghai, China).

For blockage test, the cell monolayers were pretreated with 10 μM cyclosporine A (CSA, a blocker of mitochondrial permeability transition (MPT), Sigma) for 30 min, 50 μM Z-VAD-FMK (pan-caspase inhibitor), Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhitor, Sigma) for 1 h at 37°C (Jin et al., 2009 (link); Hu et al., 2013 (link)), 25 μM LY294002 (PI3K/Akt pathway inhibitor, Sigma) for 1 h at 37°C, or 10 mM N-acetyl-cysteine (NAC, an anti-oxidant, Sigma) for 1 h at 37°C. In these tests, macrophages without blocker pretreatment were used as the controls. For RNA interference test, small interfering RNAs (siRNAs) specific to silence the mouse or human bid, aif, and endoG genes were designed and synthesized by Invitrogen Co. (Shanghai, China). The sequences of siRNAs are 5′-CGACUGUCAACUUUAUUAATT-3′ in J774A.1 and 5′-GGCCACUGUUUGGAAAUAATT-3′ in THP-1 (targeting bid); 5′-GGAAAUAUGGGAAAGAUCCTT-3′ in J774A.1 and 5′-GGCUACGUCCAGGAGCGCACC-3′ in THP-1 (targeting aif); 5′-GGACCGAGGCTGATGGGAA-3′ in J774A.1 and 5′-AAGAGCCGCGAGUCGUACGUG-3′ in THP-1 (targeting endoG). The transfection was performed using Lipofectamine-2000 (Invitrogen), according to the manufacturer's protocol.