Mirvana mirna inhibitor

JHOC-9 and OVISE cells were seeded into 6-well plates at a density that would yield 50% confluency after 24 h and were subsequently transfected with the mirVana^™ miRNA inhibitor (Thermo Fisher Scientific) that was specific for miR-9 (miR-9 inhibitor) or Anti-miR^™ miRNA Inhibitor Negative Control (anti-miR-NC; Thermo Fisher Scientific) at a final concentration of 100nM. Lipofectamine^® RNAiMAX Transfection Reagent (Thermo Fisher Scientific) was used for transfection, according to the manufacturer’s protocol.

The HuH-7 cells were seeded in 6-well plates at a concentration of 0.5 × 10⁵ cells per well (10–30% confluence) on the day before transfection. Each transfection was performed using Lipofectamine™ RNAiMAX (Invitrogen, Carlsbad, CA, USA) as per manufacturer instructions. Cells were transfected with mature miR-122-5p (mirVana™ miRNA mimic; Thermo Fisher) and antagomiR-122-5p (mirVana™ miRNA inhibitor; Thermo Fisher). The sequence of the mature miR-122-5p used in this study was 5′-UGGAGUGUGACAAUGGUGUUUG-3′, while that of the non-specific miRNA (HSS, Hokkaido, Japan) was 5′-GUAGGAGUAGUGAAAGGCC-3′.

MC3T3-E1 EGFP cells were transfected with Alexa Fluor 555-conjugated miR-143-3p inhibitor (mirVana miRNA Inhibitor, Thermo Fisher Scientific). SOVs were collected from the transfected cells 1 day after transfection. MC3T3-E1 cells were treated with the collected SOVs, and the cells were observed with a confocal microscope (Nikon A1-Si) 1 day after treatment. Autofluorescence was collected with photomultiplier-type detectors at wavelength emission windows of 452/45 nm (for the 488 nm laser to detect EGFP) and 525/50 nm (for the 488 nm laser to detect Alexa Fluor 555). Images were recorded with pixel dimensions of 0.05 µm. The step size was adjusted to 0.1 μm to create Z-stack images; 96 slices were captured using a depth of 9.6 µm. Raw imaging data were edited using NIS Elements software (Nikon).

MEPM and O9-1 cells were plated onto 96-well plates at a density of 5000 cells (MEPM cells) or 1000 cells (O9-1 cells) per well and treated with a mimic for negative control (4464061, mirVana miRNA mimic, ThermoFisher Scientific, Waltham, MA, USA) or each miRNA (4464066, mirVana miRNA mimic, ThermoFisher Scientific), or an inhibitor for negative control (4464079, mirVana miRNA mimic, ThermoFisher Scientific) or each miRNA (4464084; mirVana miRNA inhibitor, ThermoFisher Scientific), as previously described [50 (link)]. For chemical treatment, cells were plated onto 96-well plates at a density of 5000 (MEPM cells) or 1000 cells (O9-1 cells) per well and treated with either 10 μM atRA (R2625, Sigma-Aldrich), 50 μg/mL phenytoin (D4505, Sigma-Aldrich), 1 μM DEX (D4902, Sigma-Aldrich), or vehicle after 6 h of seeding cells. After 24, 48, or 72 h of the treatment, cell numbers were counted, as previously described [50 (link)].

Cells were plated onto 96-well cell culture plates at a density of 5000 cells (MELM cells) or 1000 cells (O9-1 cells) per well and treated with a mimic for negative control, miR-98-3p, miR-101a-3p, miR-101b-3p, miR-141-3p, miR-144-3p, miR-181a-5p, miR-196a-5p, miR-196b-5p, miR-200a-3p, and miR-710 (mirVana miRNA mimic, Thermo Fisher Scientific, Waltham, MA, USA), or an inhibitor for negative control, miR-181a-5p, miR-196a-5p, miR-196b-5p, and miR-710 (mirVana miRNA inhibitor, Thermo Fisher Scientific), using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific), according to manufacturer protocol (3 pmol of mimic and 0.3 µL of transfection reagent in 100 µL DMEM per well). Cell proliferation was measured using the Cell Counting Kit 8 (Dojindo Molecular Technologies, Gaithersburg, MD, USA) at 24, 48, or 72 h after each treatment (n = 6 per group).

Human primary MSC (Lonza) (0.5×10⁶) were mixed with Pre-miR miRNA Precursors miR-195, Pre-miR miRNA Precursors miR-497 or Pre-miR miRNA Precursor Negative Control (Scrambled - SCR) (Life Technologies) in an electroporation cuvette and electroporated using OPTI-MEM I (Invitrogen, Life Technologies) in a Gene Pulser Xcell Electroporation Systems (Bio-Rad) with the following conditions: voltage - 250 V, capacitance - 950 μF, resistance - 200 Ω [46 (link)].
For miR-195 antagonist experiments, 100nM anti-miR-195 (mirVana^® miRNA inhibitor, ThermoFisher Scientific) or SCR Negative Control (Anti-miR Negative Control #1, ThermoFisher Scientific) were used to transfect MSC using Lipofectamine 2000 reagent (Invitrogen), according to manufacturer's instructions.
MC3T3 cells (3×10⁶) plated in a 10-cm culture dish were transfected with 50 nM Pre-miR miRNA Precursors miR-143, Pre-miR miRNA Precursors miR-195, Pre-miR miRNA Precursors miR-497 or Pre-miR miRNA Precursor Negative Control (Scrambled - SCR) (Life Technologies) using Lipofectamine 2000 transfection reagent (Invitrogen, Life Technologies). Media without antibiotics was used.
Cells were collected after 12 hours and plated for the different assays. All Electroporations/Transfections were confirmed by RT-qPCR.