INS-1 832/13 and 828/33 cells were seeded into a 12-well plate at a density of 4×105 cells/well and treated for 48 h with miR-21 5p (accession no. MI0000569) mimic used at a concentration of 45 pmol (Qiagen, Düsseldorf, Germany), or 100 pmol locked nucleic acid (LNA) inhibitor (Exiqon, Vedbaek, Denmark), or negative controls (Qiagen, Exiqon) that had been complexed with 3 μl Lipofectamine 3000 and 100 μl Opti-MEM (both from Thermo Fisher, Grand Island, NY, USA). Relative miR-21 expression after transfection is shown in the electronic supplementary material [ESM] Fig. 1 .
For human islet dispersion and transfection, after 4 h of routine incubation at 37°C, islets were suspended in 4 ml of Accutase solution (Millipore, Billerica, MA, USA) with 100 units of DNase I (Millipore) in a thermal mixer at 37°C, 1000 rev/min for 10 min, followed by the addition of 10 ml of culture medium. Dispersed cells were collected by centrifugation at 200 g for 3 min, and then resuspended in culture medium and seeded into 12-well tissue culture plates (Falcon, Tewksbury, MA, USA). Dispersed cells from 200 islets were transfected with 23 pmol of either negative control miRNA or miR-21 mimic using Lipofectamine 3000 reagent as described above. Cleaved caspase 3 ELISA (ThermoFisher) was performed on transfected human islets as per the manufacturer’s protocol.
For human islet dispersion and transfection, after 4 h of routine incubation at 37°C, islets were suspended in 4 ml of Accutase solution (Millipore, Billerica, MA, USA) with 100 units of DNase I (Millipore) in a thermal mixer at 37°C, 1000 rev/min for 10 min, followed by the addition of 10 ml of culture medium. Dispersed cells were collected by centrifugation at 200 g for 3 min, and then resuspended in culture medium and seeded into 12-well tissue culture plates (Falcon, Tewksbury, MA, USA). Dispersed cells from 200 islets were transfected with 23 pmol of either negative control miRNA or miR-21 mimic using Lipofectamine 3000 reagent as described above. Cleaved caspase 3 ELISA (ThermoFisher) was performed on transfected human islets as per the manufacturer’s protocol.