Jetprime dna sirna transfection reagent

DU 145 and PC-3 cells with endogenous KLF5 deletion were established as previously described 35 (link). Briefly, the CRISPR-cas9 system was utilized to delete endogenous KLF5 expression following a previous published protocol 26 (link). KLF5-null clones were identified by Western blotting and confirmed by sequencing the PCR product with primers 5'-CACAATCGACAAAATAAGCCTG-3' and 5'-CAGTAGCTGGTACAGGTGGCCC-3'.
In the retroviral system, an expression vector PLHCX with or without different KLF5 variants, pMD2.G and pSPAX2, was transfected into 293T cells with the jetPRIME DNA/siRNA transfection reagent (Polyplus transfection) according to the manufacturer's instructions. Medium containing viruses was filtered at 48 hours (pore size, 0.45 μm, Corning, NY, USA). After virus infection, cells were selected with medium containing hygromycin B (ChemCruz, Dallas, TX, USA) for at least 8 days.

The MYOC gene was cloned into the pCDH-EF1α-MCS-T2A-Puro lentiviral expression plasmid. Lentivirus was packaged by using jetPRIME® DNA & siRNA Transfection Reagent (polyplus) according to manufacturer’s instructions. Lentiviral infections of THP-1 cells were performed by centrifugation at 1200g at 37 °C in 24-well plates for 2 h. Screening of cells for puromycin resistance was conducted 72 h post-transduction for 2 weeks with 5 µg·mL⁻¹ puromycin. TDMs were differentiated for 48 h in RPMI-1640 medium (10% foetal bovine serum (FBS)) containing 1% penicillin/streptomycin and 250 ng·mL⁻¹ PMA (Phorbol 12-myristate 13-acetate, PMA) (Sangon Biotech, China).

For overexpression of CPT2 in CRC cells, the full-length CPT2 cDNA was amplified from HCT116 cell and then inserted into pcDNA3.1 vector. RiboBio (Guangzhou, China) was approached to purchase Small interfering RNAs (siRNAs) targeting CPT2 for the purpose to silence CPT2. The following was the sequence of siRNAs targeting CPT2: CPT2 si#1: GTAGCACTGCCGCATTCAA, CPT2 si#2: GACCCTGGTTTGATATGTA. Small interfering RNAs (siRNA) transfection was carried out from the jetPRIME DNA & siRNA Transfection Reagent (PolyPlus-transfection, France). The cells were collected after 48 hours of transfection and western blot analysis was used to confirm the knockdown efficacy of the cells and was used for subsequent tests.