Dharmafect 1

NIH 3T3 cells were purchased from ATCC and cultured in Dulbecco's Minimum Essential Media (high glucose), (Hyclone) supplemented with 4 mM l-Glutamine, 1 mM Sodium Pyruvate, and 10% BCS (CO serum company). Cells were transfected with 0.2 μl/well (96 well) Dharmafect I (Thermofisher Scientific) as per the manufacturers' instructions. Cells were harvested 48 h after transfection, and gene expression was analyzed using qPCR (Life Tech).
A549 cells (ATCC® CCL-185™) were maintained in F-12K Medium (ATCC® Catalog No. 30-2004) with 10% FBS and kept in a 37°C incubator with a 5% CO₂ air atmosphere. Cells were transfected with 0.2 μl/well Dharmafect I (Thermofisher Scientific) as per the manufacturers' protocol. TGF-β was added at the time of transfection. Cells were harvested 24 and 48 h post-transfection, and RNA expression was analyzed using qPCR (Life Technologies).
LL 29 (AnHa) (ATCC ® CCL-134™) cells were maintained in Ham's F12K medium with 15% FBS and kept in a 37°C incubator with a 5% CO₂ air atmosphere. Cells were transfected with 0.2 μl/well Dharmafect I (Thermofisher Scientific) as per the manufacturers' protocol. TGF-β was added at the time of transfection. Cells were harvested 24 and 48 h post-transfection, and RNA expression was analyzed using qPCR (Life Technologies).
Col1a1 Sense Strand: 5′- GCAAGACAGUCAUCGAAUA Col1a1 Antisense Strand: 3′- CGUUCUGUCAGUAGCUUAU

INS1 cells were transfected with miR-146a-5p miRIDIAN mimics (#C-300630-03, Thermo Scientific) or a negative control miR mimic (#CN-001000-01, Thermo Scientific) using DharmaFECT 1 (Thermo Scientific) as transfection reagent. All transfections had a final oligo concentration of 30 nmol/l. Cell medium was changed at the day of transfection to medium without antibiotics. In two separate tubes, OptiMEM (Invitrogen) was mixed with DharmaFECT-1 reagent and with synthetic oligos, and the two tubes were incubated for 5 min at room temperature. The contents of the two tubes were mixed 1:1 and incubated at room temperature for 20 min. The transfection solution was then diluted 1:5 with RPMI-1640 (Invitrogen) and added to the cells. The transfection medium was removed after overnight incubation at 37°C in a humidified atmosphere of 5% CO₂. The cells were then incubated for at least another 24 h. Transfection efficiency was estimated to ~50% by co-transfection with a GFP expressing plasmid pMAX (Lonza). Furthermore, the expression level of the transfected miR mimic was examined as a transfection control. For evaluation of miR overexpression in combination with cytokine stimulation, cells were pre-treated with miR mimics for 48 h and then exposed to IL-1β (160 pg/ml) for 30 min or 6 h.