Mirna mimics and inhibitors

For the gain and loss of function assays, we used miRNA mimics and inhibitors (Exiqon) for miRNA-21 from Rattus norvegicus. First, DRG explants were transfected with lipofectamine 2000 (Thermo Fisher) with the miRNA-21 mimic 1 day after plating the DRG, and axonal elongation was quantified 4 days post-transfection. For the loss-of-function assay, SCs were transfected with lipofectamine 2000 with 50 nM LNA-enhanced antisense miRNA inhibitor of miRNA-21 or a negative control A inhibitor (YI00199006) (Exiqon) in Optimem media (Gibco). The media was replaced after 4 h with SC media. Then, EVs were purified by ultracentrifugation from SCs incubated with or without 50 μM ATP after 8 h, and DRG explants were treated daily with 0.5 μg of these EVs. Axonal elongation was measured on day 4 after treatment.

To determine the impact of candidate miRNAs in modulating the expression of Nrf2, candidate miRNAs were either overexpressed or inhibited using chemically synthetized miRNA mimics and inhibitors (Exiqon, Denmark), respectively. For this, 100 nM miRNA mimic, inhibitor, or the corresponding negative control (NC) were transfected in sub-confluent granulosa cells using Lipofectamin^® 2000 (Invitrogen, Carlsbad, CA, USA) transfection reagent in Opti-MEM I reduced-serum medium (Invitrogen, Carlsbad, CA, USA). Twenty-four hours post-transfection, cells were subjected to mRNA and protein expression analysis. Moreover, detection of intracellular ROS level, cell proliferation assay and assessment of mitochondrial activity were performed.
In order to cross-validate the regulatory role of candidate miRNAs on Nrf2 gene functions, targeted knockdown of Nrf2 was performed using bovine specific siRNA (Exiqon, Denmark). For this, sub-confluent granulosa cells were transfected with 200 nM of siRNA -Nrf2 or siRNA negative control (NC) using Lipofectamin 2000 in Opti-MEM I reduced-serum medium. Twenty-four hours post-transfection, the cells were subjected to intracellular ROS level detection, cell proliferation assay, assessment of mitochondrial activity, mRNA and protein expression pattern analyses.