Entranster r4000

Plasmids sh-OPN and sh-NC, as well as miR-34c and NCmimics were designed and synthesized by Wuhan Viraltherapy Technologies Co., Ltd. A549 and H460 cells were inoculated into Petri dishes one day in advance. On the second day, 1 μg of shRNA or miRNA mimics and 3 μL of Entranster-R4000 (EnGreen Biosystem, Beijing, China) were diluted into 50 μL with the serum-free medium. After standing for 10 min at indoor temperature, they were mixed into transfection complexes and added to cells that had been replaced with fresh medium. The transfection efficiency was assessed by Western blot. Subsequent experiments were performed 48 h after transfection.

The following BC168687 siRNA and negative control siRNA sequences were synthesized (Novobio Scientific, Inc., Shanghai, China) and used: BC168687-rat-159 (5′-GAGAUUAUUAAGGUGUACUTT-3′), BC168687-rat-1172 (5′-GACGGUUGAUACUGACUCUTT-3′), BC168687-rat-2400 (5′-GUUGGAUCCUUCUCAAUCATT-3′) and negative control siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′). The three different BC168687 siRNA duplexes were transfected into SGCs using the Entranster^™-R4000 (Engreen Biosystem, Ltd.) according to the manufacturer's protocol. After 48 h, the expression levels of BC168687 were evaluated by reverse transcription quantitative-polymerase chain reaction (RT-qPCR).

A total of 1×10⁶ HCT116 cells/well were transfected with miR-223 mimics or NC using Entranster™-R4000 (Engreen Biosystem, Ltd.), then transfected with 1 µg pTriEx-FBXW7 overexpression plasmid or empty plasmid 12 h later. The cells were collected 48 h later and lysed for protein detection by western blotting. The cell viability was also detected at the indicated times following miR-223 transfection and apoptosis rates were detected by flow cytometry at 48 h post-transfection.

The human HCT116 cell line was purchased from American Type Culture Collection and cultured in high-glucose DMEM (Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) in a 37°C incubator with 5% CO₂. Cells in the logarithmic growth phase (80% confluence) were used for experiments. The 50 ng pMIR-REPORT luciferase reporter plasmids (Ambion; 50 ng), miR-223 mimics (5′-UGUCAGUUUGUCAAAUACCCCA-3′), miR mimic control (miCtr, 5′-UUCUCCGAACGUGUCACGUTT-3′), miR-223 inhibitor (5′-UGGGGUAUUUGACAAACUGACA-3′) and miR-223 inhibitor control (Ctr; 5′-CAGUACUUUUGUGUAGUACAA-3′) were transfected using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Small interfering RNA (siRNA) targeting FBXW7 (siFBXW7−1, 5′-ACAGGACAGUGUUUACAAA-3′; siFBXW7−2, 5′-CCAUGCAAAGUCUCAGAAU-3′) and negative control (si-NC, 5′-UUCUCCGAACGUGUCACGUTT-3′) were transfected using Entranster™-R4000 (Engreen Biosystem, Ltd.), according to the manufacturer's instructions. All small nucleic acids were purchased from Shanghai GenePharma Co., Ltd. and were used at a final concentration of 20 nM. The cells were treated or collected at the indicated times after transfection.