Jetprime dna

Cells were transfected with validated siRNA for EZH2 at a concentration of 100 nM using jetPRIME DNA and siRNA Transfection Reagent (Polyplus-transfection SA, NY, USA). The EZH2 gene target-specific siRNAs were purchased from Gene Pharma (Shanghai, China) and the targeting sequences are listed in Supplementary Table S2. For the qRT-PCR analyses of mRNAs, total RNA was isolated using TRIZOL and reverse transcribed to cDNA using the PrimeScript 1st Strand cDNA Synthesis kit. The qRT-PCR reactions were performed using SYBR Green technology and the PCR primers are listed in Supplementary Table S2.

Mel290-EV and Mel290-PRAME cells (1×10⁶ cells per sample) were transfected with pDR-GFP (Addgene plasmid # 26475) using jetPRIME DNA and/or siRNA transfection reagent (Polyplus) and selected for puromycin resistance (2 μg/ml). Upon 60% confluence, the cells were transfected with a plasmid expressing the restriction enzyme I-SceI (pCBASce1) (Addgene plasmid # 26477) using jetPRIME DNA and/or siRNA transfection reagent (Polyplus). This restriction enzyme cuts the reporter plasmid and when repaired by HR, GFP is expressed ^{30 (link)}. Ninety-six hours after I-SceI transfection, cells were harvested, centrifuged at 300 g for 5 minutes at 4°C, washed with cold 1x PBS and resuspended in cold 1x PBS. Flow cytometry and data analysis were performed by the University of Miami Sylvester Comprehensive Cancer Center Flow Cytometry Shared Resource (FCSR).

The two sequences for porcine Occludin gene knockdown were 5′- ggaagactggatcagggaata-3′ and 5′-gcaggagtacaagagcttaca-3′. The two sequences for porcine Zo 1 gene were 5′-ggatggcgctacaagtgatga-3′ and 5′-ggactcctctggaatgcatca-3′. The two sequences for porcine Claudin 2 gene knockdown were 5′-tctcctcgttggcctgtat-3′ and 5′-gcatcatttcctccctgtt-3′. The scrambled control siRNA sequence was 5′-ggatccttgacaataccaa-3′. The siRNA double-stranded oligonucleotides sequences were cloned into pLSLG lentiviral vector. The respective lentiviral vectors and packaging vectors were transfected into HEK 293FT cells. After 2 days, the virus was collected and PIECs were infected. Four days later, the infected cells were used for experiments as described.
The two sequences for porcine P38 gene knockdown were 5′-ggcaagaaactacattcaa-3′ and 5′-ggcccggcatacagatgat-3′. The siP38 oligos and control oligo were transfected into PIECs using jetPrime® DNA and siRNA Transfection Reagent (Polyplus transfection, Illkirch, France) according to the instructions. After 48 h, the transfected cells were used for experiments.