Pcdna3.1 hygromycin plasmid vector

Total RNA was obtained from astrocytoma cell line 1321N1 using TRIzol and reversetranscribed with Transcriptor Reverse Transcriptase according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). Full-length human PFKFB3-4 was generated by standard PCR using primers PFKFB3-4 reverse and PFKFB3-4 forward (Table S2). The resulting amplicon was cloned into pcDNA3.1/Hygromycin plasmid vector (Invitrogen, Waltham, MA, USA) using ApaI and AflII. Similarly, the full-length fragment of human PFKFB3-5 was amplified with primers PFKFB3-5 reverse and PFKFB3-5 forward and subcloned into the plasmid pGEM-T using the T/A Cloning Kit (Promega, Mannheim, Germany). Subsequently, the amplicon was digested with XbaI and HindIII and inserted into the pcDNA3.1/Hygromycin plasmid vector (Invitrogen, Waltham, MA, USA). After confirmation by sequencing and enzymatic digest, both constructs were assigned the names pcDNA-PFKFB3-4 and pcDNA-PFKFB3-5.