Endofectin max transfection reagent

Expression plasmids for the miR-196a-5p mimic control, miR-196a-5p mimic, miR-196a-5p inhibitor control and miR-196a-5p inhibitor were purchased from Fulengene. For further study, including flow cytometry, reverse transcription-quantitative (RT-q) PCR and Western blotting, cells were seeded into 6-well plates (Corning Inc.) at a concentration of 5x10⁵/well; for MTT assays, the cells were seeded into 96-well plates at 4x10³/well. When the cells had reached ~80% confluence, each expression plasmid was mixed with Opti-MEM^TM I (Invitrogen; Thermo Fisher Scientific, Inc.) and incubated for 5 min. Next, EndoFectin^TM-Max transfection reagent (GeneCopoeia, Inc.) was diluted in Opti-MEM^TM I and incubated for 5 min. The plasmids were then added to the diluted transfection reagent to form a transfection complex, which was then incubated for 10 min. Finally, the mixtures were added to each well and incubated in a humidified cell incubator with an atmosphere of 5% CO₂ at 37°C for 48–72 h. The cells were then collected, and transfection efficiency was confirmed by RT-qPCR and Western blotting. All experiments were carried out at room temperature.

The siRNAs were purchased from GeneBio (GeneBio, China). 1×10⁶ MCF7 and SK-BR-3 cells were, respectively, seeded in each well of a six-well culture plate (KIRGEN, USA). 7.5µL of siRNA combined with 7.5µL of EndoFectin^TM-Max transfection reagent (GeneCopoeia, USA) were diluted in 250µL MEM medium (Gibco, USA) respectively, and incubated for 5 min. The mixture was then used for transient interference assay following the manufacturer’s protocol. The sequences of siRNAs are as follows:
5ʹ-UUCUCCGAACGUGUCACGUTT-3ʹ (siN.C.)
5ʹ-GCAUUAGUACCUACAUAUUTT-3ʹ (siPPEF1)

The human breast cancer cell lines, including MCF-7, BT-474, SK-BR-3, and MD-MBA-231, along with skin fibroblast cells and esophageal squamous cell carcinoma cell lines, KYSE170 and TE,1 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) or RPMI 1640 (Gibco, USA) medium supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 50 units of penicillin, and 50 µg/mL of streptomycin. All cells were cultured under the standard conditions of 5% CO₂ at 37 °C. For transfection, cells were transfected with the indicated combinations of JARID2-silencing RNA [human JARID2 small interfering RNA (siRNA)] using EndoFectinTM-Max transfection reagent (GeneCopoeia, Guangzhou, China) according to the manufacturer’s instructions. Briefly, 1 day before transfection, 1.5×10⁵ cells were plated on a 6-well plate in 2 mL medium free of antibiotics. The next day, transfection reagents containing 0.1 nmol of siRNA and 5 µL of Lipofectamine RNAiMAX in a final volume of 260 µL with Opti-MEM I were added to each well and incubated for 24 hours prior to further assay. A non-targeting sequence was used as a negative control.

AML12 mouse hepatocytes (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM/F12 with 10% FBS supplemented with 1% Insulin-Transferrin-Selenium (ITS) (51500056; Invitrogen, Waltham, MA, USA), 40 ng/mL dexamethasone, and 1% penicillin streptomycin mixture. The plRES2-EGFP-NC and plRES2-EGFP-Hamp plasmids were transfected into AML12 cells using the EndoFectin™ Max transfection reagent (GeneCopoeia, Rockville, MD, USA) following the manufacturer’s instructions. Briefly, AML12 cells (5×10⁴ per well) were seeded into a 12-well plate. After 24 h, the cells were transfected with either 2 or 4 µg of the plasmids using the EndoFectin™ Max transfection reagent.