Extraction of total RNA from indicated tumor tissues or cell lines was done using TRIzol reagent (Takara). Employing 1 μg of total RNA and PrimeScript RT Reagent Kit (Takara), the first-strand cDNA was synthesized. The levels of miRNA were determined by qRT-PCR with SYBR-Green ΙΙ PCR kit (Takara) and U6 snRNA was used as the reference. LncRNA and mRNA concentrations were measured employing SYBR-Green ΙΙ PCR kit (Takara). β-actin was used as the internal control. Supplementary Table S2 shows the sequences of the primers used.
Extraction of protein was from the HCC cells was done using RIPA buffer, and determined using the BCA Protein Assay Kit (Beyotime Biotechnology). Separation of extracted proteins (40 μg per lane) was done by SDS-polyacrylamide gel electrophoresis and the separated proteins were transferred to polyvinylidenedifluoride (PVDF) membranes (Bio-Rad). The PVDF membranes were blocked using 5% non-fat milk for 1 h and primary antibodies against GLUT1 (Biorbyt, St Louis, MO, USA; orb157188), HK2 (Abcam; ab227198), LDHA (Abcam; ab52488), ALDH1A3 (Proteintech; 29,373–1-AP), β-actin (Abcam; ab8226), Alix (Abcam; ab88743), CD9 (Abcam; ab236630) and TSG101 (Abcam; ab125011) were used. Secondary antibodies were labeled with Peroxidase. The ECL detection system (Thermo) was used for visualization.
Extraction of protein was from the HCC cells was done using RIPA buffer, and determined using the BCA Protein Assay Kit (Beyotime Biotechnology). Separation of extracted proteins (40 μg per lane) was done by SDS-polyacrylamide gel electrophoresis and the separated proteins were transferred to polyvinylidenedifluoride (PVDF) membranes (Bio-Rad). The PVDF membranes were blocked using 5% non-fat milk for 1 h and primary antibodies against GLUT1 (Biorbyt, St Louis, MO, USA; orb157188), HK2 (Abcam; ab227198), LDHA (Abcam; ab52488), ALDH1A3 (Proteintech; 29,373–1-AP), β-actin (Abcam; ab8226), Alix (Abcam; ab88743), CD9 (Abcam; ab236630) and TSG101 (Abcam; ab125011) were used. Secondary antibodies were labeled with Peroxidase. The ECL detection system (Thermo) was used for visualization.