Rox plus

50 mg of cryo-preserved tissue was homogenized, and the total RNA was isolated using the mirVana miRNA Isolation Kit (Ambion, Foster City, CA, USA) as described previously [17 ,18 (link)] (KIRC N = 103, NAT N = 20). The DNA elimination was achieved by treatment of the RNA with DNase (DNA-free Kit, Ambion).
The RNA of the cell lines was isolated from pellets using the Total RNA Purification Mini Spin Column Kit (Genaxxon bioscience GmbH, Ulm, DE). The RNA quantity and quality were determined using the NanoDrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
Reverse transcription of approximately 1 μg total RNA was performed using the PrimeScript RT Reagent Kit with gDNA Eraser. The DONSON-knockdown validation PCR experiments were carried out following genomic DNA elimination by treating 1 μg of total RNA with gDNA Eraser for 2 min at 42 °C and subsequent reverse transcription with the same PrimeScript RT Reagent Kit. 5 ng of the resulting cDNA was used for real-time PCR (1× SYBR Premix Ex Taq II with ROX Plus and 10 pmol/μl PCR primers; all reagents: Takara Bio, Saint-Germain-en-Laye, France).
The following primer sequences were used: DONSON forward: 5′-GTCCAGCATTGTAGGGCAAC-3′ and reverse: 5′-GGCTCTGCTGGAAGGTACAA-3′, β-Actin forward: 5′-CCAACCGCGAGAAGATGA-3′ and reverse: 5′-CCAGAGGCGTACAGGGATAG-3′. The primer annealing temperature was 60 °C for both primer pairs.

Total RNA of the cocultured cells was extracted with TRIzol reagent (Gibco BRL, USA) according to the manufacturer's instructions after 1, 6, and 24 h, and the purity and concentration of the sample were determined on a spectrophotometer. cDNA was then synthesized using 2 μg total RNA with a PrimeScript RT Reagent Kit (Qiagen, Hilden, Germany) following the manufacturer's specifications. Primer sequences were synthesized by Invitrogen (Carlsbad, CA, USA) [Table 1]. Real-time polymerase chain reaction (RT-PCR) was then carried out with SYBR^® Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa Bio, Dalian, China) and ROX plus (TaKaRa Bio) on an ABI 7500 system (Applied Biosystems, Foster City, CA, USA). Multiple changes of mRNA expression were calculated using the 2^-ΔΔct method, where Δ ct is the difference between the genes TLR2 and β-Actin, and ΔΔct for the sample = Δct of the actual sample − Δct of the lowest expression sample.[12 (link)13 (link)]

TRNzol (#DP424) was acquired from Tiangen Biotech Co., Ltd. (Beijing, China); PrimeScript RT reagent Kit with gDNA Eraser (#RR047B), SYBR Premix Ex Taq II (Tli RNaseH Plus), and ROX plus (#RR82LR) were acquired from Takara Bio (Tokyo, Japan); an methyl thiazolyl tetrazolium (MTT) assay kit, bicinchoninic acid (BCA) protein quantification kit, and a NanoDrop 2000 spectrophotometer were acquired from Thermo Fisher Scientific (Waltham, MA, USA); a RIPA Total Protein Extraction Kit was acquired from Sigma-Aldrich (St. Louis, MI, USA); a mitochondria extract kit, enzyme-linked immunosorbent assay (ELISA) kits, and mitochondrial membrane potential JC-1 kit were acquired from Solarbio Science & Technology (Beijing, China); an adenosine triphosphate (ATP) kit, superoxide dismutase (SOD) kit, and methane dicarboxylic aldehyde (MDA) kit were acquired from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); a FACSCalibur II Flow Cytometer was acquired from BD Biosciences (Franklin Lakes, NJ, USA); an XDS-2B inverted fluorescence microscope was acquired from Chongqing Optical & Electrical Instrument Co., Ltd. (Chongqing, China); and an ABI 7500 real-time PCR instrument was acquired from Applied Biosystems (Thermo Fisher Scientific); the autophagy inhibitor 3-methyladenine (3MA) was purchased from Selleck Chemicals (Houston, TX, USA).