Lipofectamine 3000 transfection agent

SiRNAs, miRNA mimics and inhibitors were purchased from GenePharma (Shanghai, China). The oligonucleotides of miRNA mimics, inhibitors and siRNAs were shown in Table 2. Human SSC line was seeded at 1 x 10⁵/cm² density and cultured in DMEM/F12 with 10% FBS overnight.
Transfection of miRNA mimics or inhibitor and siRNAs was performed using lipofectamine 3000 transfection agent (Life Technologies, Carlsbad, CA, USA). After 48 hr of culture, cells were harvested for subsequent analyses.
The lentivirus, namely PGMLV-CMV-MCS-EF1-ZsGreen1, was purchased from Jiman Biotechnology CO., LTD (Shanghai, China). Human SSC line was plated at a density of 5 × 10⁵ cells in 100 mm plates, and these cells were cultured with DMEM/F12 and 10% FBS overnight. Culture medium was changed with fresh DMEM/F12 containing 10⁸ TU/ml PGMLV-CMV-MCS-EF1-ZsGreen1 and 10 μg/ml polybrene, and the cells were incubated at 37° C in 5% CO₂ overnight. After 24 hr of culture, the medium was changed with fresh DMEM/F12 and 10% FBS, and the expression of ZsGreen1 was detected under a fluorescence microscope (Nikon Eclipse Ti-S, Nikon Corporation, Tokyo, Japan). The infected cells were cultured and expanded in DMEM/F12 medium supplemented with 10% FBS and 1% antibiotic containing penicillin and streptomycin (Gibco).

Mouse microglia cell line BV-2 was obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China). The cells were cultured in dulbecco’s modified eagle medium (DMEM, Gbico) supplemented with 10% Fetal bovine serum (FBS, Biological Industries) and 1% penicillin/streptomycin (P/S, Servicebio) at 37 °C in a 5% CO₂ incubator. Culture medium changed once every two days. In order to explore the varying expression of RORα in vitro, BV2 cells were seeded into 6-well plates at the density of 5 × 10⁵/ml and treated with different concentrations of MPP⁺ (0,10,20,50 μM, Sigma, D048) for a duration of 24 h. In order to verify the neuroprotective effect, MLT (50 μM), SR3335 (10 μM), SR1078 (5 μM) was used for pre-treatment half an hour before MPP⁺ (50 μM) intervention. Constant volume of phosphatic buffer solution (PBS) or DMSO was used as control. SiRNA-mediated knockdown of RORα was conducted using Lipofectamine 3000 transfection agent (Invitrogen) according to the manufacturer’s protocol. All the samples were collected 24 h after MPP⁺ treatment for following experiments.

Human MSCs cultured in ODM were transfected with miR-130a mimics or inhibitor, miR-27b mimics or inhibitor and its negative controls (Applied Biosystems/Ambion, Austin, TX, United States) using the Lipofectamine 3000 transfection agent (Invitrogen, Carlsbad, CA, United States). The alkaline phosphatase activity was measured using an assay kit (Colorimetric; Abcam). At indicated times, the conditioned medium was collected. Into a 96-well plate, 80 μl of samples was added followed by the addition of 50 μl of a 5 mM pNPP solution to each well. The reaction mixture was incubated at 25°C for 60 min and the reaction was stopped by adding 20 μl of the stop solution followed by gently shaking of the plate. The p-nitrophenol product, which was generated via enzymatic hydrolysis of the p-nitrophenylphosphate substrate, was detected at OD 405 nm using a microplate reader synergy HTX Multi-Mode Reader (BioTek Instruments, VT, United States).

miR34a mimic or inhibitor and negative control were purchased from Ruibo Biology Technology Company (Ruibo, Guangzhou, China). HEI-OC1 cells were plated into 6-well plates at a density of 1.5 × 10⁵/well. Cells were grown to 50% confluence and then transfected with 5 µM miR34a mimic or 10 µM inhibitor using serum-free Opti-MEM (Gibco, USA) and Lipofectamine 3000 transfection agent (Invitrogen, USA) following the manufacturer’s instructions. Negative controls were generated using mimic or inhibitor control with the same procedure. Cells were incubated with the transfection mixture for 8 h at 37 °C, then replaced with normal DMEM and further incubated for 40 h.