Quantitative real-time PCR

Total RNA was extracted from cells using a NucleoSpin RNA kit (Macherery-Nagel, # 740955) following the recommendations of the supplier. After synthesis of cDNA with the aid of SuperScript VILO^TM Master Mix (Invitrogen, #11755250), quantitative real-time PCR was conducted to quantify target gene expression. The specific primers used were as follows: PD-L1 (Forward 5ʹ-TGCCGACTACAAGCGAATTACTG-3ʹ, Reverse 5ʹ-CTGCTTGTCCAGATGACTTCGG-3ʹ), PDK1 (Forward 5ʹ-CCAAGACCTCGTGTTGAGACC-3ʹ, Reverse 5ʹ-AATACAGCTTCAGGTCTCCTTGG-3ʹ) PFKP (Forward 5ʹ-CGGAAGTTCCTGGAGCACCTCTC-3ʹ, Reverse 5ʹ-AAGTACACCTTGGCCCCCACGTA-3ʹ), PFKFB3 (Forward 5ʹ- CAGTTGTGGCCTCCAATATC-3ʹ, Reverse 5ʹ-GCTTCATAGCAACTGATCC-3ʹ) and GAPDH (Forward 5ʹ-TCCATGACAACTTTGGTATCGTG-3ʹ Reverse 5ʹ-ACAGTCTTCTGGGTGGCAGTG-3ʹ). JNK1-specific primers were purchased from Santa Cruz (Santa Cruz, #sc-29380). GAPDH was employed as the internal reference gene.