Jetprime versatile dna sirna transfection reagent

Small-interfering RNAs (siRNAs) were purchased from Thermo Fisher Scientific. Cells were transfected with siRNA duplexes using the jetPRIME Versatile DNA/siRNA transfection reagent (Polyplus, New York, NY, USA) according to the manufacturer’s instructions. The sense and antisense strands of Cripto-1 siRNA were as follows: si-Cripto1#1: 5′-GGA UCA UGG CCA UUU CUA AAG UCU U-3′ (sense) and 5′-AAG ACU UUA GAA AUG GCC AUG AUC C-3′ (antisense); si-Cripto1#2: 5′-UCA UGC AAA UUU CAU GAC CAG UAA A-3′ (sense) and 5′-UUU ACU GGU CAU GAA AUU UGC AUG A-3′ (antisense); si-Cripto1#3: 5′-GGG CCA UCA GGA AUU UGC UCG UCC A-3′ (sense) and 5′-UGG ACG AGC AAA UUC CUG AUG GCC C-3′ (antisense).

The HEK293T cells were cultured in DMEM supplemented with 10% FBS and seeded into 48-well cell culture plates at a density of 2 × 10⁴ cells/well. They were then incubated overnight at 37°C with 5% CO₂. Once the cells reached a confluency of 70–80%, the recombinant plasmid pCAGGS-gE was transfected into the cells using the jetPRIME^® Versatile DNA/siRNA transfection reagent (Polyplus, France) and cultured at 37°C for 48 h. After culturing, the plates were fixed with methanol (precooled to −20°C) for 20 min at room temperature (RT). Next, the plates were washed with PBST and incubated with skim milk (5%) at 37°C for 1 h. Then, anti-PRV gE was used as a primary antibody for 30 min, while DMEM served as a negative control. Subsequently, the plates were washed three times with PBST and incubated with anti-mouse IgG H&L (FITC) (Solarbio, Beijing, China) for half an hour at 37°C. Cells were simultaneously stained using DAPI (4′,6-diamidino-2-phenylindole) from Solarbio. After the final washing, fluorescence signals were visualized using fluorescence microscopy from ZEISS in Jena, Germany.