Cfx connect detector instrument

Total RNA of osteosarcoma patients’ specimens and different cell lines was extracted with Trizol reagent (Vazyme, Cat# R401-01, China), and cDNA was obtained by reverse transcription process using Hifair II Strand cDNA Synthesis Super Mix (Cat# 11120ES60, Yeasen, Shanghai, China) based on the product instructions. RT-qPCR was carried out with the CFX Connect Detector instrument (Bio-Rad, USA) and the relative mRNA expression level was calculated by the 2^−ΔΔCt method [16 (link)]. The primer sequences of genes were presented in Table S2.

The negative control and PDK-1 overexpression plasmids were synthesized by Shanghai Jikai Company (China). 143B and HOS cells were transfected with plasmids using GP-Transfect-Mate (GenePharma, Suzhou, China) following the manufacturer’s protocol. After 48 h, total RNA was extracted with Trizol reagent (Vazyme, Cat# R401-01, China) based on the product, and then RNA was measured using a spectrometer (NanoDrop Technologies, USA). After cDNA synthesis, RT-qPCR was carried out with a CFX Connect Detector instrument (Bio-Rad, USA) and the relative mRNA expression levels were calculated by the 2^−ΔΔCt method [30 (link)]. The following primers were used:
GAPDH-F: 5′-GGAGTCCACTGGCGTCTTCA-3′
GAPDH-R: 5′-GTCATGAGTCCTTCCACGATACC-3′
PDK-1-F: 5′- GAGGAAGCAGGAAGGATCAGT-3′
PDK-1-R: 5′- GAACGGATGGTGTCCTGAGA-3′