Superrt cdna kit

A total of 1.2 μg of RNA was used for cDNA synthesis using the SuperRT cDNA Kit (CWBIO, China). Quantitative real-time PCR (qPCR) was performed on a QuantStudio 12 K Flex Real-time PCR System (Applied Biosystems, USA). The ribosomal protein S3 (rpS3) gene was used as the reference gene [97 (link)]. The qPCR conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Three biological replicates were performed for each treatment, and each biological replicate was carried out in triplicate. The expression levels of genes were calculated using the 2^−ΔΔct method, and One-way ANOVA with Tukey's post hoc test was used for statistical analysis. The primers used in the qPCR are described in Additional file 10: Table S10.

Total RNA was isolated from frozen xenograft tumor specimens in all treated groups using the Trizol Reagent (Invitrogen, Grand Island, NY, USA). The cDNA was produced by Super RT cDNA kit (CWBio, Beijing, China) following the instructions. RT-PCR was performed using RealMasterMix (SYBR Green, TIANGEN, Beijing, China). Relative gene expression was established according to the 2^(−ΔΔCT) method.
The tumor tissues of all groups were dislocated by sonication and extracted at 4 °C for 30 min. Whole-cell protein lysates were separated by electrophoresis through a 12% SDS-PAGE gels, and then, proteins were transported onto a polyvinylidene fluoride membrane (PVDF). The membrane was incubated with primary antibodies including DR5 (1:50,000, Abcam, Cambridge, MA, USA), DR4 (1:1000, Abcam) and GAPDH (1:2000, ZSGB-BIO, Beijing, China). Primary antibodies were exposed using goat anti-rabbit horseradish peroxidase-link secondary antibodies (1:5000, SANTA CRUZ BIOTECHNOLOGY, Dallas, TX, USA). The detection of specific proteins was exposed to Kodak-X-Omat films carried out using ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

Virus RNA was extracted from allantoic fluid using the RNeasy Mini kit (Qiagen, Hilden, Germany) and was transcribed into complimentary DNA (cDNA) using the Uni12 primer (5′-AGC AAA AGC AGG-3′) and SuperRT cDNA kit (CWBIO, Beijing, China) according to the manufacturers' recommendations. cDNAs of eight segments were then amplified with gene specific primers designed by the Chinese National Influenza Center (primer sequences are available upon request). The PCR reaction contained 2 μL cDNA, 1 μL forward primer and reverse primer, 12.5 μL 2 × EsTaq MasterMix (CWBIO, Beijing, China) and 8.5 μL RNase-free water with a final volume of 25 μL. A single PCR program was used for all primers, i.e., initial denaturation at 94 °C for 2 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min and 30 s.

N-Acetyl-1-cysteine (NAC, purity ≥ 98%) and patulin (PAT, purity ≥ 99%) were supplied by Sigma-Aldrich (St. Louis, MO, USA). The LDH-assay kit, ROS assay kit, total SOD assay kit, annexin V–FITC apoptosis assay kit, Hoechst 33342 dyes, GSH and GSSG assay kit, GR assay kit, total GPx assay kit, ATP assay kit, mitochondrial membrane potential (MMP) assay kit, the assay kits of caspase 3, 8, and 9, and BCA protein-assay kit were obtained from Beyotime Institute of Biotechnology (Beijing, China). MitoSOX Red Mitochondrial Superoxide Indicator was purchased from Invitrogen Corporation (St. Louis, MO, USA). The CAT test kit was obtained from Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). The Ultrapure RNA kit, Super RT cDNA kit, and UltraSYBR mixture were purchased from CWBIO (Beijing, China).