Lipofectamine 2000 reagent kit

Cells were assigned to three groups: HMGA2-siRNA group, mock-siRNA group and
non-transfected control group (HMGA2-siRNA and mock-siRNA; Jima Pharmaceutical
Technology, Shanghai, China). RNAi was applied for transient transfection in
ACHN cells. Transient cell transfection was implemented according to the
instructions of the Lipofectamine® 2000 reagent kit (Thermo Fisher Scientific,
Rockford, IL, USA). Briefly, two high-pressure sterilized Eppendorf tubes were
used; and 10 µl siRNA + 240 µl serum-free RPMI-1640 medium and 10 µl
Lipofectamine® + 240 µl serum-free RPMI-1640 were added to each tube,
respectively. After gentle mixing, these tubes were incubated at room
temperature for 20 min to form an siRNA-Lipofectamine® complex. The mixed
liposomes and siRNA solution were added to each well dropwise and cultured in an
incubator. Then, the RNA was extracted after 24 h for subsequent reverse
transcription quantitative real-time polymerase chain reaction (RT-qPCR) (Thermo
Fisher Scientific) analysis as described below. The protein was extracted after
48 h for Western blot analysis as described below.

An in vitro transfection assay was performed according to the Lipofectamine 2000 reagent kit (Invitrogen) instructions, and the expression of the oipDNA in MDCK cells was detected using an indirect immunofluorescence test. The transfection experiment was divided into 4 groups of MDCK cells. Group 1 was MDCK cells transfected with pβH9N1SH5/DGL NPs containing 4 μg of pβH9N1SH5; Group 2 was MDCK cells transfected with a commercial transfection reagent mixed with 4 μg of pβH9N1SH5; Group 3 was MDCK cells transfected with naked pβH9N1SH5 as a negative control; and Group 4 was untransfected MDCK cells as a control. AIV-positive serum (Shanghai Veterinary Research Institute, China) and fluorescein isothiocyanate-labeled goat anti-mouse IgG (Sigma, St. Louis, MO) were diluted at 1:100 and 1:5, respectively. Epifluorescence images were obtained using an Axio observer Z1 microscope (Carl Zeiss AG, Oberkochen, Germany).

One-day-old, cell monolayers exhibiting 70% to 80% confluency in six-well plates were used for recombinant plasmid transfection. Briefly, the plasmid and liposome complexes were prepared according to the recommended ratio of the Lipofectamine™ 2000 Reagent kit (Invitrogen, USA) with 1 μg DNA per 2.5 μL of liposomes. Both 4 μg of plasmid DNA and 10 μL of Lipofectamine^TM 2000 were separately diluted into 100 μL OPTI-MEM medium (Invitrogen) and maintained at room temperature for 5 min, before gently mixing. The plasmid/liposome complexes were incubated at room temperature for 20 min. Then, 200 μL of the plasmid/liposome mixture was diluted in 800 μL of the OPTI-MEM medium, and subsequently injected into the well. The six-well plates were cultured in 5% CO₂ at 37°C, and 6 h after transfection, the cultural medium was replaced by DMEM supplemented with 2% FBS.