Reverse transcription system kit

Total RNA was extracted from cancer and non-cancerous tissues using Trizol (Invitrogen, USA) according to the instructions of the Reverse Transcription System kit (Vazyme Biotech, China). Reverse transcription was performed according to kit instructions followed by quantitative PCR using the Roche 480 II Real-Time PCR machine, and the real time PCR conditions and reaction mixture composition were showed in Tables 6 and 7. All the gene expression levels were analyzed and calculated by using the 2-ΔΔCt method [29 (link)]. NF1 and β-Actin primers were purchased from Sangon Biotech Company. The primer sequences for NF1 were: forward, 5′-ACACATGCAAAATGGGAACA-3′ and reverse, 5′-TGGGACATTCGCCTCTTAAC-3′. The primer sequences for β-Actin were: forward, 5′-AGCGAGCATCCCCCAAAGTT-3′ and reverse, 5′-GGGCACGAAGGCTCATCATT-3′.

Specifically, total RNA was extracted from liver tissues and spleen T lymphocytes by TRIzol reagent (#257401, Invitrogen, United States). RNA was reverse transcribed in to cDNA by reverse transcription system kit (#R222-01, Vazyme, China). The mRNA levels were measured by qPCR using SYBR Green qPCR Master Mix (#Q111-02/03, Vazyme, China) according to the manufacturer protocol. The primers were listed in Supplementary Table S2. Gene expression was normalized to expression of β-actin.

The FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, China,RC112–01) was used to extract total RNA from tissues and cells according to the manufacturer’s instructions for RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR). A reverse-transcription system kit (Vazyme, China, R223–01) was used to complete the reverse transcription of lincRNA and mRNA, which was then evaluated by qPCR using a Universal SYBR qPCR Master Mix kit (Vazyme, China, Q711–02) Use GAPDH as an internal control to determine the mRNA level, according to the operation handbook. For relative quantification, the 2Δ-CT approach was applied. Three independent duplicates of each qRT-PCR experiment were carried out.

After CNE1 cells treatment with CuB (1,10,50 nM) for 24 h, total RNAs were extracted using Trizol reagent (Invitrogen, USA) and dissolved in nuclease-free water. 1 µg RNA was used to reverse transcribe cDNA using Reverse Transcription system Kit (Nanjing Vazyme Biotech Co., Ltd., China) as described in the manufactures’ protocol. cDNA was used for quantitative Real-Time PCR on the QuantStudio 7 Flex (ThermoFisher, MA) using GPX4(fwd: 5′-ACAAGAACGGCTGCGTGGTGAA-3′; rev: 5′-GCCACACACTTGTGGAGCTAGA-3′) or actin (fwd: 5′-ATCTGGCACCACACCTTCTAC-3′; rev: 5′-CAGGTCCAGACGCAGGATG-3′) primers in a SYBR green reaction to determine mRNA levels. GPX4 mRNA expression levels were normalized to the expression of actin mRNA.

TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from human tissues or cells according to the manufacturer’s instructions. A reverse transcription system kit (Vazyme Biotech Co., Ltd., Shanghai, China) was used to generate the first strand of cDNA. The ChamQ Universal SYBR qPCR Master Mix and ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) were used for qPCR. The qPCR parameters were as follows: 40 cycles of 30 s at 95 °C, then 10 s at 95 °C, and 30 s at 60 °C. GAPDH was used as an endogenous control (Supplementary Table S3). The relative fold change was analyzed by the 2-ΔΔC(t) method. Each experiment was performed in triplicate.

An RNA-prep Pure Tissue Kit (Nanjing Vazyme Biotech, Nanjing, China) was used to extract total RNA from the PSCs. First-strand cDNA was synthesized from total RNA using a reverse transcription system kit (Nanjing Vazyme Biotech). PCR was then used to amplify the LEL coding sequence from the cDNA using a sense primer (5′- CGC GGA TCC ATG TTT CCA GCA CCG CTT CAA G-3′) comprising a BamHI site (underlined) and an antisense primer (5′-CCG CTC GAG TCA TTC ATA GTT TTT CAA GGA G-3′) comprising a XhoL I site (underlined). The PCR amplicons were ligated into the pET32a (+) plasmid (Novagen, Darmstadt, Germany) and transformed into Escherichia coli BL21 (DE3) cells (Tiangen, Beijing, China). Isopropyl-1-thio-β-D-galactopyranoside (1 mM; IPTG) was used to induce expression from the plasmid for 6 h at 37°C. Inclusion bodies were obtained from the E. coli cells, suspended in lysis buffer containing 8 M urea, and incubated for 2.5 h on ice to completely solubilize the recombinant protein. Ni²⁺ affinity chromatography with a His-affinity resin column (Bio-Rad, Hercules, CA, United States) was used to purify the His-tagged rEg-TSP11 protein, which was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A NanoDrop 2000c (Bio-Rad) instrument was used to determine protein concentration.