Transit x2 dynamic delivery system mir6000

The RNA interfering and plasmid transfection assays were performed in BC cells and conducted as previously reported [24 (link), 36 (link), 38 (link)]. The siRNAs and shRNAs were purchased from RiboBio and GeneChem (Shanghai, China), respectively. The siRNA and shRNA sequences are listed in Table S4.
For siRNAs and plasmids transfection, the transfection mixture was prepared with siRNAs or plasmids, RPMI opti-MEM (Gibco), and TransIT-X2® Dynamic Delivery System (MIR 6000, Mirus Bio) according to the system in the User Guide of MIR 6000. The transfection mixture was then added to plates where cells had been seeded for 24 h.
For lentivirus transfection, lentivirus containing shRNAs targeting Importin-7 or control shRNAs were generated. Firstly, a complete medium containing polybrene (5 μg/ml) was used to incubate the BC cells for 15 min, and lentivirus was then added into plates for 48 h. Medium containing puromycin (2 μg/ml) was used to eliminate the unsuccessfully infected cells.

H9c2 cells (1 × 10⁶ cells/ml) were seeded in a 6-well plate containing a complete culture medium and cultured to 70–80% confluence before transfection. For knockdown of Beclin-1/Becn1 (Coiled-Coil, Moesin-Like BCL2-Interacting Protein), 10 μl of transfection reagent (TransIT-X2^® Dynamic Delivery System # MIR6000, Mirus) was diluted in 250 μl OptiMEM serum reduced medium, to this 25 nM of Beclin-1 siRNA was added and incubated at room temperature for 30 min to form transfection reagent: siRNA complex development. The transfection reagent: siRNA complex was then added to 70–80% confluence H9c2 cells (replaced with culture medium supplemented with 1%FBS) and incubated at 37°C with 5% CO2 in an incubator for 1 h. After 1 h incubation, culture medium FBS was increased to 10% (complete culture medium), and these pre- Beclin-1- siRNA transfected cells were used for mimicking hypoxic conditions. siRNA and transfection reagents concentrations were used according to the manufacturer’s instructions.

The highly purified plasmids pBlue-HPRS/JL3-1, pBlue-HPRS103 and pBlue-HPRS103-N123I were introduced respectively into DF-1 cells using the TransIT-X2 Dynamic Delivery system (MIR6000, Mirus Bio LLC, USA). The culture supernatant containing the virus stocks was harvested 7 days later and then blind-passaged into secondary DF-1 cells. The rescued viruses were named rHPRS/JL3-1, rHPRS103, and rHPRS103-N123I. The titer of the rescued recombinant virus was determined based on the TCID₅₀ value using the Reed and Muench method [64 (link)].