Block it lentiviral rnai expression system

Galectin-9-specific siRNA oligomers (siGal-9; catalog number E-011319-00-0005) and scrambled oligomers (siCon; catalog number D-001950-01-05) were purchased from Dharmacon (GE Healthcare, Lafayette, CO, USA). siRNA transfection was performed according to manufacturer's instruction. Briefly, 5×10³ MSCs were seeded onto a well of a 96-well plate. The next day, adherent MSCs were transfected with siGal-9 or siCon. The cells were further stimulated with 10 ng/ml TNF-α and 20 ng/ml INF-γ for 48 h. After knockdown of galectin-9 was confirmed by western blotting, the cells were subjected to in vitro immunosuppression assay and ELISA. For lentiviral shorthairpin RNA (shRNA)-mediated gene knockdown, BLOCK-iT Lentiviral RNAi Expression System was used purchased from Invitrogen. All processes including lentiviral cloning, packaging, production, and infection were performed according to manufacturer's instruction. Target sequences in human galectin-9 (NCBI reference NM_009587.2) are as follows; 5'-GGACTTCAGATCACTGT-3' and 5'-GGAAG ACACACATGCCTTTCC-3'.

We knocked down the FAP gene (Genbank accession No. U09278) in CAFs isolated from human and mouse GC tumor tissue using BLOCK-iT™ Lentiviral RNAi Expression System (Invitrogen™, Carlsbad, CA, USA). All the procedures followed the manufacturer’s instruction. The sequences of the double-stranded oligonucleotide (ds oligo) used to construct pENTR™/U6 vector (Invitrogen™) were 5′-CACCGGTCGCCTGTTGGGAGTAAATCGAAATTTACTCCCAACAGGCGACC-3′ (“top strand” for human FAP); 5′-AAAAGGTCGCCTGTTGGGAGTAAATTTCGATTTACTCCCAACAGGCGACC-3′ (“bottom strand” for human FAP); 5′-CACCGCATAAAGCTTGGCTGTTTCTCGAAAGAAACAGCCAAGCTTTATGC-3′ (“top strand for mouse FAP”); 5′-AAAAGCATAAAGCTTGGCTGTTTCTTTCGAGAAACAGCC AAGCTTTATGC-3′ (“bottom strand” for mouse FAP). These oligonucleotides were annealed to make double-stranded oligonucleotides with a 5′ overhang (CACC) and a 3′ overhang (AAAA). Expression vectors were kept in One Shot^® Stbl3™ Chemically Competent E. coli. Lentivirus was produced in 293FT cells.

AC005224.4 shRNA or scrambled shRNA was constructed into BLOCK-iT™ lentiviral RNAi expression system (Invitrogen), which was designed to suppress AC005224.4 production in SKOV3 cells. Briefly, SKOV3 cells (3 × 10⁵ cells/dish) were seeded onto 35-mm dishes. Next, 1 day after seeding, cultures were added with lentiviral particles (200 μL) in a 2-mL RPMI-1640 medium containing 10% FBS, followed by 24-h incubation (5% CO₂, 37 °C). The cells were successfully infected by lentiviral particles (Lv-sh-NC or Lv-sh-AC005224.4) for 48 h. After that, the cells were harvested and subjected to qRT-PCR analysis to determine the efficiency of lentiviral particles in suppressing AC005224.4 expression.