Glut1

Total cellular RNA was extracted from 0.5 to 1 × 10⁶ cells using TRIzol (Life Technologies; Darmstadt, Germany) (36 (link)). The primers were purchased from QIAGEN, including Glut-1 (QT01044953; QIAGEN, Germany), HK2 (QIAGEN; QT00155582), and LDHα (QIAGEN; QT02325414). Reverse transcription was performed by using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Germany). RNA quantitation was performed using SYBR green and a StepOne Plus RT-PCR system according to the manufacturer's instructions (QIAGEN, Hilden, Germany).
Western blot analysis was performed according to the established protocol (37 (link)). Whole-cell lysates were performed using cell lysis buffer (Thermo Fisher Scientific, Germany). Equal amounts of protein (100 μg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Germany). After blocking the non-specific sites with 5% BSA, specific antibodies including phospho-Akt (Cell Signaling, Germany), phospho-mTOR (Cell Signaling, Germany) and HK2 (Cell Signaling, Germany) were incubated with membranes. Immunoreactive bands were then developed with an enhanced chemiluminescence system (GE Healthcare, Germany).

Total RNA was extracted with the RNeasy Mini Kit (Qiagen, USA) following the manufacturer’s instructions and cDNA was synthesized using the SuperScript III First-Strand Synthesis SuperMix (Invitrogen Life Technologies, USA). RT-PCR was performed using CFX384 Real Time System (Bio-Rad, USA) and SYBR Green Master Mix (Applied Biosystems Foster City, CA). Gene expression was then measured with CFX384 Real Time System (Bio-Rad, USA) with primer sets for endothelin-1 (EDN1, Catalog #QT00088235, Qiagen, USA), erythropoietin (EPO, forward: 5′-GCT GCA TGT GGA TAA AGC CCG-3′; reverse: 5′-CAC ACC TGG TCA TCT GTC CC-3′) (Sigma-Aldrich, USA), glucose transporter 1 (GLUT1, Catalog #QT00068957 Qiagen, USA), vascular endothelial growth factor (VEGF, Catalog #QT01682072 Qiagen, USA), Bax forward: 5′-GTT TCA TCC AGG ATC GAG CAG-3′; reverse: 5′-CAT CTT CTT CCA GAT GGT GA-3′ and (Sigma-Aldrich, USA) and Bcl2 forward: 5′-CCT GTG GAT GAC TGA GTA CC-3′; reverse: 5′-GAG ACA GCC AGG AGA AAT CA-3′ (Sigma-Aldrich, USA) with β-Actin as internal control (Sigma-Aldrich, USA) and using Power SYBR Green PCR Master Mix (Applied Biosystems Foster City, CA).