Transfected or untransfected A549 and H446 cells were treated with or without 50 µM shikonin (Shanghai Yuanye Biotechnology Co., Ltd.) for 24 h at 37°C. Total RNA was extracted from clinical specimens or cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA using a PrimeScript RT kit (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol. qPCR was subsequently performed on an ABI 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using a SYBR® Premix Ex Taq™ kit (Takara Biotechnology Co., Ltd.). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 3 min; followed by 40 cycles at 95°C for 5 sec and 60°C for 30 sec. The following primers pairs (Sangon Biotech Co., Ltd.) were used for the qPCR: PFKFB2 forward, 5′-GCTGCTTGGTGGGAGTGATAA-3′ and reverse, 5′-TGAGAAGCCAAGTGTCAGGG-3′; and β-actin forward, 5′-GAGGACCCTGGATGTGACAG-3′ and reverse, 5′-AAGACCTGTACGCCAACACA-3′. Relative mRNA expression levels were quantified using the 2−ΔΔCq method (27) and normalized to β-actin.

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