Lipofectamin 2000

Immortalized MFN2^−/− homozygous knockout MEFs (Chen et al, 2003) (link) were cultured at 37°C and 5% CO₂ in a humidified incubator in DMEM–GlutaMAX containing 4.5 g/l glucose (#61965026; Thermo Fisher Scientific) supplemented with 1 mM sodium pyruvate (#11360039; Thermo Fisher Scientific), 100 μM nonessential amino acids (#11140035; Thermo Fisher Scientific), and 10% FBS (S0115; Biochrom). The cells were transiently transfected using Lipofectamin 2000 (#11668; Thermo Fisher Scientific). Lipofectamin 2000 was incubated 5 min at RT in Opti-MEM (#31985070; Thermo Fisher Scientific) before adding 1 μg plasmid per six-well plate and incubation for 15 min at RT. Transfection mix was added drop wise to plated cells. Transient transfection was performed for 48 h, whereby the medium was exchanged after 24 h.

The knockdown of Drosha was performed using siRNA technology (FlexiTube, Qiagen). Two different siRNAs for Drosha (Mn_Ethohi2_1 #SI00996499 and Mn_Ethohi2_3 #SI00996513) or negative control siRNA (#1027280) were transfected with Lipofectamin®2000 (Thermo Scientific). For Atg5 and Smn knockdown, we transfected siRNAs (Atg5 s62452; Smn s133926 or negative control 4390843, Silencer® Select Pre-designed siRNA, Thermo Scientific) with Lipofectamin®2000. To inhibit miR-218 activity, miR-218 inhibitor (AM10328, Thermo Scientific) and control (AM17010, Thermo Scientific) were transfected with Lipofectamine®3000 (Thermo Scientific). For transfection of shRNA containing plasmid, we used 1 ug of DNA with 2 ug of Lipofectamin®3000 per coverslip. We mostly followed the manufacturer’s instructions, however, we replaced the culture media 1 hour after transfection for WT motor neurons, while we changed the media 45 min after transfection for SMA motor neurons.

HeLa and Cos7 cells were transfected with siRNA using Oligofectamin (Life Technologies) according to the manufacturer’s protocol. SK-MEL-2 cells were transfected with siRNA using siRNA Max (Life Technologies) according to the manufacturer’s protocol. To achieve optimal knockdown efficiency, two rounds of silencing were performed on day 1 and day 2. For transient overexpression of proteins in knockdown cells, plasmids were transfected together with the second round of siRNA using Lipofectamin2000 (Life Technologies) according to the manufacturer’s protocol. Cells were analysed 72 h after the first siRNA transfection. For transient overexpression of proteins in untreated cells, plasmids were transfected 24 h prior to analysis using Lipofectamin2000 (Life Technologies).

Cells were divided into four groups: blank group (without any transfected sequence), NC group (transfected with negative control sequence 5′-UUCUCCGAACGUGUCACGUTT-3′), miR-34a mimics group (transfected with miR-34a mimics sequence 5′-ACCGUCACAGAAUCGACCAACA-3′), and miR-34a inhibitors group (transfected with miR-34a inhibitor sequence 5′-ACAACCAGCUAAGACACUGCCA-3′). All oligonucleotide sequences were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Huh 7 cells in logarithmic phase of growth were inoculated into 6-well-plate. When the cells reached 30% to 50% confluence in complete medium without antibiotic, they were transfected according to the instruction of lipofectamin 2000 (Invitrogen Inc., Carlsbad, CA, USA). The siRNA plasmid (final concentration: 50 nM) and 5 μl lipofectamin 2000 were diluted by 250 μl serum-free Opti-MEM (Gibco, Invitrogen, Carlsbad, CA, USA) through even mixture, respectively, and were then incubated for 5 min at room temperature. The two diluted solutions were mixed gently, incubated for 20 min at room temperature, added into cell-contained well, and incubated at 37°C incubator with 5% CO₂. After 6 to 8 h, the culture solution was changed, the complete medium was added and the cells were incubated for 24 to 48 h for following experiments.

HEK293T cells (ATCC-CRL-11268, American-type culture collection, United States) were grown onto 60-mm diameter culture dishes for 24 h in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% of fetal bovine serum (FBS), in a humidified atmosphere at 95% air and 5% CO₂. Cells were transfected using Lipofectamin 2000 (Invitrogen Life Technologies) following provider’s instructions (1 µg DNA/3 µl Lipofectamin 2000). 24 h later, cells were transferred to 24-well plates at 150,000 cells/well and incubated for 24 h. Cells were incubated for 30 min at 37 °C with 10 µM forskolin in presence or absence of allatostatin and/or antagonist (B5 or B6) in 0.5 ml DMEM. The cells were then washed with phosphate-buffered saline and lysed. The intracellular levels of cyclic adenosine monophosphate (cAMP) were determined with a DetectX® Cyclic AMP (cAMP) Direct Immunoassay kit (K019-H1; Arbor Assays, Ann Arbor, MI, USA) according to the manufacturer instructions.