Tp800

Total RNA was extracted using Sepasol (Nacalai Tesque) according to the manufacturer’s protocol. cDNA was generated using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO) according to the manufacturer’s protocol. Quantification of mRNA was performed with a Light Cycler 480 (Roche) or TP-800 (TAKARA) using THUNDERBIRD SYBR qPCR Mix (TOYOBO). Primer sets used for qRT-PCR analysis were CCL5 forward: 5′-ACC​ACA​CCC​TGC​TGC​TTT​G-3′, CCL5 reverse: 5′-CAC​ACA​CTT​GGC​GGT​TCT​TTC-3′, SOD2 forward: 5′-TGG​AAG​CCA​TCA​AAC​GTG​AC-3′, SOD2 reverse: 5′-AAA​CCA​AGC​CAA​CCC​CAA​C-3′, HO1 forward: 5′-CTT​TCA​GAA​GGG​CCA​GGT​G-3′, HO1 reverse: 5′-GGA​AGT​AGA​CAG​GGG​CGA​AG-3′, and actin forward: 5′-TCC​CTG​GAG​AAG​AGC​TAC​GAG-3′, actin reverse: 5′-GGA​AGG​AAG​GCT​GGA​AGA​GTG-3′.

The extraction of RNA and quantitative real-time PCR (qRT-PCR) was performed as described previously [24 (link)]. For BAG3 qRT-PCR analysis, the following primers were used: forward, 5’-ATGCGCGATTCCGAACTGAG-3′ and reverse, 5’-AGGATGAGCAGTCAGAGGCAG-3′; additionally, 18S rRNA was used as an endogenous control: forward, 5’-AATAGCCTTTGCCATCAC-3′ and reverse, 5’-CGTTCCACCTCATCCTC-3′. BAG3 mRNA expression was analyzed using a real-time PCR instrument, TP800 (Takara, Japan). The relative BAG3 expression was normalized to 18S rRNA, and data analyses were performed using the comparative CT method [25 (link)].

Treated cells were lysed in plates after removing the medium. Total RNAs were obtained using Trizol Reagent (Thermo, Waltham, MA, USA). cDNAs were prepared with random hexamers from mRNA, using AMV reverse-transcription kit (Promega). The sequence of primers was as follows: GAPDH: forward 5′-gaaggtgaaggtcggagtc-3′, reverse 5′-gaagatggtgatgggatttc-3′; mTOR: forward 5′-gaggtgtggtttgaccgaag-3′, reverse 5′-atcaggttggatgggtgtct-3′; eIF4E: forward 5′-gttatcagtcccacgcagac-3′, reverse 5′-gaagaggctttggttcagc-3′; 4EBP: forward 5′-ctccttgtgcctccactgat-3′, reverse 5′-gggttcgttcttgtccactt-3′. cDNA amplification reactions were performed using PowerSYBR Green PCR master mix (Thermo). Quantitative PCR reactions were performed on the iQ5™ (BIO-RAD, Hercules, CA, USA). Data were analyzed by a TP800 (Takara, Dalian, China). The 2-ΔΔC_t method for relative quantization was used to determine mRNA expression. Fold change was determined as 2-ΔΔC_t and target mRNA expression was normalized to GAPDH.

Quantitative real-time reverse transcription (RT) PCR was performed using SYBR Premix Ex Taq (Takara Bio Inc., Otsu, Shiga, Japan). The following primer was used: Rad51 (forward 5′-gcataaatgccaacgatgtg-3′, reverse 5′-atgatctctgaccgcctttg-3′). For quantification, all values were normalized to β-actin using the ΔΔCt method with data from 3–5 independent experiments. Data were analyzed using a TP800 (Takara Bio Inc., Otsu, Shiga, Japan).

Total RNA was isolated from indicated cells using Trizol reagent (Invitrogen) and Ultrapure RNA Kit from CWBIO (Beijing, China) according to the manufacturer’s instructions. 0.8 microgram of total RNA was reverse-transcribed using M-MLV reverse transcriptase (Promega). Quantitative real-time PCR was performed on a real-time PCR machine TP800 (Takara) using SYBR master mixture (Takara). The primer sequences are as follows: RRS1 forward, 5′- CCCTACCGGACACCAGAGTAA-3′, RRS1 reverse, 5′- CCGAAAAGGGGTTGAAACTTCC-3′; and GAPDH forward, 5′- TGACTTCAACAGCGACACCCA-3′, GAPDH reverse, 5′- CACCCTGTTGCTGTAGCCAAA-3. The relative RRS1 expression was normalized to GAPDH, and data analysis was conducted using the comparative CT method.