Opti mem

A panel of siRNA’s against candidate ion channels and controls was purchased from Dharmacon (GE Dharmacon) each siRNA was a pool of 4 sequences (Supplementary Table S3). All siRNA was reconstituted to 20 µM stock concentration in RNase-free water. C-20/A4 cells were plated at a density of 250,000 cells/well of a 6 well plate in 10% FCS media as described above, and incubated at 37 °C and 5% CO₂ for 30 minutes. Reconstituted siRNA was diluted to 20 nM working concentration in 200 μl of Opti-MEM (Gibco). 12 µl of HiPerFect (Qiagen) was added to the siRNA and Opti-MEM and incubated for 15 minutes at room temperature. Following incubation, siRNA solution was added to cells in a dropwise manner and incubated in normal culture conditions for 48 h. 12 h before collection, media was changed on all wells and replaced with 1% FCS media. siPiezo1 and siPiezo2 of the same pooled sequences was purchased from Dharmacon for further experiments. C-20/A4 cells were transfected with 20 nM siRNA against Piezo1, Piezo2 and a Scrambled control in the presence of HiPerFect, and cultured for 72 h before treatment with CP-154526 (50 µM). Phase contrast images were collected at 5 h post-CP treatment.

T98G cells were seeded into 96 well plates at 1,000 cells per well in 80μl of medium. 24 hours after seeding, cells were transfected with 885 Human miScript miRNA mimics (Qiagen, Valencia, CA) in 96 well plates. Transfection was performed by the addition of a transfection mix consisting of 15.5μl of Opti-MEM, 0.5μl of Hiperfect (Qiagen), and 4μl of 2μM siRNA. 48 hours after transfection, miRNA cytotoxicity was scored by visual inspection under the microscope, Qiagen cell death siRNA used as a positive control for comparison on each 96 well plate. miRNAs causing >50% reduction in cell number were excluded from further consideration. Cell extracts were prepared from the 96 plates as described previously [43 (link)].

Cells were transfected using 6 µL interferin (Polyplus transfection, Illkirch, France) and 200 µL optimem (Thermo Fisher Scientific, Waltham, MA, USA) 24 h post-seeding (6-well plates), following manufacturer’s instructions. For miRNA overexpression, cells were transfected with miRIDIAN microRNA miR-22-3p mimic or miRIDIAN microRNA Mimic Negative Control #1 (Horizon Discovery, Cambridge, UK) with concentrations ranging between 1 and 20 nM depending on the assay. For gene silencing, 10 nM of control siRNA (AllStars Negative Control siRNA, Qiagen, Hilden, Germany) or siRNA against thrombospondin 1 (siTSP1, Hs_THBS1_3 FlexiTube siRNA, Qiagen, Hilden, Germany) was transfected, also using interferin and optimem, following manufacturer’s instructions.

Total RNA from H‐VLNs or GF‐VLNs or miRNA mimics were transfected in BMDMs using Lipofectamine RNAiMAX (Invitrogen) per manufacturer's protocol. Briefly, 2 μl of Lipofectamine RNAiMAX were diluted in Opti‐MEM (ThermoFisher Scientific) and added to 20 nM of miRNA mimic (Qiagen) or AllStars negative control siRNA (Qiagen) in Opti‐MEM. After 20 min of incubation, the mixture was added to the BMDMs in a 24‐well plate. After 24 h of transfection, the cells were treated with LPS + ATP to activate the NLRP3 inflammasome. 400 ng/ml of total RNAs from H‐VLNs or GF‐VLNs were transfected in BMDMs using 2 μl of Lipofectamine RNAiMAX. For the dose experiment, different amounts of GF‐VLN RNAs were added to H‐VLN RNAs to ensure the total transfected RNAs at the concentration of 400 ng/ml. Alternatively, different amounts of negative control miRNA were added to miR‐4057 to ensure the total transfected RNAs at the concentration of 20 nM.

Cells were cultured in 12-well plates until 70–80% confluency. 100 µl/ml OptiMEM, 9 µl/ml Hyperfect (Qiagen, Hilden, Germany), and 3 µl siRNA (20 µM siRNA-Control (Qiagen, #1027310) or siRNA-p54nrb#2 (Qiagen, #2999579)) were mixed, incubated for 15 min at room temperature and added dropwise to the cells. Then the cells were incubated in 5% CO₂ at 37 °C.

Three types of specific siRNAs targeting human MET and a scrambled (sc) siRNA were synthesized by Bioneer (Daejeon, Korea) (sequences in Supplementary Table S1). Hs746T and H1993 cells seeded in 6-well plates and grown to 60–70% confluency were transfected with siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in an Opti-MEM (Qiagen, Germantown, MD, USA). After 6 h, the medium was replaced with complete medium and the cells were harvested 48 h after transfection.

We screened miRNAs that modify microglial differentiation and activation using the miRCURY LNA^TM microRNA inhibitor library (Exiqon, Vedbaek, Denmark). A total of 739 miRNA inhibitors were screened. miRNA inhibitors were suspended in Opti-MEM and HiperFect transfection reagent (Qiagen, Hilden, Germany) and were incubated for 15 min at room temperature. Then, miRNA transfectants were added to LN⁻ cell-astrocyte co-culture seeded on 96-well plates at a final concentration of 40 nM on days 0 and 3. On day 7, wells were observed microscopically by two authors (RS and HS) in a blinded manner and morphological findings were independently assessed in a semi-quantitative manner. Wells were scored as hits when both investigators judged that the number of SR cells changed significantly.