Control small interfering si rna

PC12 cells were obtained from American Type Culture Collection, cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin, and maintained at 37°C in a humidified atmosphere with 5% CO₂.
PC12 cells (1×10⁶ cell/ml) were transfected with 100 nM miR-138-5p inhibitor (5′-CGGCCUGATTCACAACACCAGCT-3′; Shanghai GenePharma Co., Ltd.), 100 nM inhibitor control (5′-CAGUACUUUUGUGUAGUACAA-3′; Shanghai GenePharma Co., Ltd.), 0.2 µM control-small interfering (si)RNA (cat. no. Sc-36869; Santa Cruz Biotechnology, Inc.), 0.2 µM SIRT1-siRNA (cat. no. Sc-40986; Santa Cruz Biotechnology, Inc.) or 100 nM miR-138-5p inhibitor + 0.2 µM SIRT1-siRNA using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The efficiency of cell transfection was evaluated via reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis at 48 h after transfection.
An in vitro cell model of SCI in PC12 cells was established according to a previous study (33 (link)). In brief, PC12 cells were subjected to lipopolysaccharide (LPS; 100 ng/ml) for 4 h at 37°C. Control cells were left untreated.

ARPE-19 cells were plated into 6-well plates and incubated for 24 h before transfection. Cells were transfected with 100 nM inhibitor control (5′-GCCUCCGGCUUCGCACCUCU-3′; Shanghai GenePharma Co., Ltd., Shanghai, China), 100 nM miR-217 inhibitor (antagomir; 5′-UACUGCAUCAGGAACUGAUUGGA-3′; Shanghai GenePharma Co., Ltd.), 0.2 µM control-small interfering (si)RNA (cat. no. sc-36869; Santa Cruz Biotechnology, Inc.), 0.2 µM SIRT1-siRNA (cat. no. sc-40986; Santa Cruz Biotechnology, Inc.), or 100 nM miR-217 inhibitor + 0.2 µM SIRT1-siRNA using Lipofectamine 2000 reagent for 24 h. Then, we performed RT-qPCR assay to detect the transfection efficiency.

In total, 100 nM miR-24-3p inhibitor (antagonist of miR-24-3p; 5'-CUGUUCCUGCUGAACUGAGCCA-3'), 100 nM inhibitor control (5'-CAGUACUUUUGUGUAGUACAA-3'), 100 nM miR-24-3p mimic (sense: 5'-UGGCUCAGUUCAGCAGGAACAG-3'; anti-sense: 5'-GUUCCUGCUGAACUGAGCCAUU-3), 100 nM mimic control (sense: 5'-UUCUCCGAACGUGUCACGUTT-3'; anti-sense: 5'-ACGUGACACGUUCGGAGAATT-3'; all from Shanghai GenePharma Co., Ltd.), 1 µM control small interfering (si)RNA (cat. no. sc-36869; Santa Cruz Biotechnology, Inc.), 0.2 µM Bcl-2L11-siRNA (cat. no. sc-29802; Santa Cruz Biotechnology, Inc.), 100 nM miR-24-3p inhibitor + 1 µM control-siRNA, or 100 nM miR-24-3p inhibitor + 0.2 µM Bcl-2L11-siRNA were transfected into VSMCs using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for in accordance with the manufacturer's protocol. Reverse transcription-quantitative (RT-q)PCR was performed to detect the efficiency of cell transfection 48 h after incubation, at 37˚C.

Ectopic ESCs were seeded into 6-well plates at a density of 5x10⁴ cells/ml and cultured at 37˚C overnight. A total of 50 nM inhibitor control (5'-GGACCAAATCTCGAGATTTGG-3'; Shanghai GenePharma, Co., Ltd.), 50 nM miR-139-5p inhibitor (5'-ACTGGAGACACGTGCACTGTAGA-3'; Shanghai GenePharma, Co., Ltd.), 1 µM control-small interfering (si)RNA (cat. no. sc-36869; Santa Cruz Biotechnology, Inc.), 1 µM BBC3-siRNA (cat. no. sc-37153; Santa Cruz Biotechnology, Inc.), 50 nM miR-139-5p inhibitor + 1 µM control-siRNA or 50 nM miR-139-5p inhibitor + 1 µM BBC3-siRNA were transfected into ectopic ESCs using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Following 48 h of transfection at 37˚C, cells were collected (centrifugation at 4˚C at 12,000 x g for 15 min) and the transfection efficiency was determined using RT-qPCR. Subsequent experiments were performed 48 h after transfection.