Amaxa nucleofector apparatus

To transfect RNA oligos into DRG neurons, the dissociated neurons from lumbar DRGs were centrifuged to remove the supernatant and resuspended in 100 μL of Amaxa electroporation buffer for mouse neuron (Lonza Cologne GmbH, Cologne, Germany) with siRNAs (0.2 nmol per transfection). siFgf3 (sense: CAGAGACCUUGGUACGUGUtt; Antisense: ACACGUACCAAGGUCUCUGgg). Suspended cells were then transferred to a 2.0 mm cuvette and nucleofected with the Amaxa Nucleofector apparatus. After electroporation, cells were immediately mixed to the desired volume of prewarmed culture medium and plated on precoated coverslips or culture dishes. After neurons fully attached to the coverslips or culture dishes (4–6 hr), the culture medium was changed to remove the remnant electroporation buffer.

siRNA-mediated knockdown was performed as previously described^{38 (link)}. In brief, Raw264.7 macrophages or BMDMs were harvested and re-suspended in 90 µL of solution V (for RAW264.7 cells; Lonza) or solution for mouse macrophages (for BMDMs; Lonza). Scrambled siRNAs or siRNAs against Kxd1 or Plekhm2 (Dhamacon; 1.5 µg/reaction) were added to the cell suspension, transferred to an electroporation cuvette and nucleofected using the Amaxa Nucleofector apparatus (Amaxa Biosystems) with program D-032 (for RAW264.7 macrophages) or Y-001 (for BMDMs). At 24 h after transfection, cells were harvested and plated for assays.

Dami or CMK cells were transiently transfected with plasmids encoding S6K1-WT or the S6K1-T389E, S6K1-T389A and S6K1-D3E mutants as described elsewhere [7] (link). Briefly, Dami or CMK cells were washed in cold phosphate-buffered saline and resuspended in the specified electroporation buffer to a final concentration of 1.2×10⁸ cells/ml. Two micrograms of each of the mutant plasmids (described above) was mixed with 0.1 ml of cell suspension. The mixture was transferred to a 2.0-mm electroporation cuvette and nucleofected with an Amaxa Nucleofector™ apparatus (Amaxa, Cologne, Germany). After transfection, the cells were resuspended in RPMI 1640 medium with 10% FCS and incubated overnight. After incubation, the transfected Dami or CMK cells were induced with SP600125 (32 µM) for 72 hours in a humidified atmosphere of 5% CO₂ at 37°C after pretreatment with or without U0126 (10 µM) and LY294002 (30 µM) for 1 hour. Dami or CMK cells that were not transfected with mutant plasmids but were induced with SP600125 were used as controls. After a 72-hour incubation, the cells were fixed and prepared for flow cytometric analysis as described above.

In brief, THP-1 cells were harvested and re-suspended in 90 µL of human monocyte nucleofector solution (VPA-1007; Lonza). Scrambled siRNAs or siRNAs against IL-36RN (Dhamacon; 1.5 µg/reaction) were then added to the cells and the cell suspension was transferred into an electroporation cuvet and nucleofected using the Amaxa Nucleofector apparatus (Amaxa Biosystems) with programme Y-001, as previously described [44 (link)]. Cells were harvested and plated for assays at 24 hr after transfection.

Primary cortical neurons were prepared from brains of P0 C57 mice as described previously [28 (link)]. Before plating, neurons suspended in transfection medium (200 μl) were mixed with 1 μg pCAG-EGFP and 3 μg shRNA for immunocytochemistry. Then, neurons were transferred into a 2.0-mm electroporation cuvette (Fisher) and transfected by electroporation using the Amaxa Nucleofector apparatus.

Based on the highest level of expression of BART6-3p, in vitro studies using mimic and inhibitor sequences of BART6-3p were performed in Akata cells. Briefly, transient transfections of the Akata cell line were performed using an Amaxa nucleofector apparatus (Amaxa, Cologne-Germany), program G23 and transfection solution V according to the manufacturer’s instructions. 5×10⁶ cells were transfected with 100 nM of BART6-3p inhibitor (Custom synthesized by Dharmacon- Thermo Scientific, Germany), 10 nM of negative control of miRNA inhibitor (I-300145-01; Dharmacon-Thermo Scientific, Germany) or the transfection solution as a mock. Transfection efficiency was assessed using transfecting 2 μg of pmaxGFP and detecting both fluorescence and cell viability by flow cytometry. Cells were harvested 24 hours after transfection for RNA extraction and qRT-PCR and 48 hours post-transfection for protein extraction and western blotting.