Mir 17

Total RNA was extracted from sorted bone marrow derived macrophages, lung AM and brain microglia of WT or Csf1r^CreDicer^fl/fl knockout mice with miRNeasy Mini Kit (QIAGEN). The RNA was reverse-transcribed to cDNA with miRCURY LNA^TM microRNA RT Kit (Exiqon). Quantitative real-time PCR reactions were prepared using FastStart Universal SYBR Green Master (ROX, Roche) and carried out in QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). The U6 primer set was purchased from Exiqon (Product No.: 203907). All other miRNA primer sets were purchased from Life Technologies, including miR-17 (Assay ID: 002308), miR-18a (Assay ID: 002422), miR-19a (Assay ID: 000395), miR-19b (Assay ID: 000396), miR-20a (Assay ID: 000580), miR-92a (Assay ID: 000430), miR-21 (Assay ID: 000397), miR-146a (Assay ID: 000468), miR-150 (Assay ID: 000473), miR-155 (Assay ID: 002571) and miR-233 (Assay ID: 002295). Relative miRNA expressions were normalized to U6 expression.

Luciferase reporter construct encompassing the 3’UTR of ASK1 mRNA (NM_005923) with the binding sites for miR-17 and empty control reporter construct were obtained from Genecopia (Rockville, MD). ASK1 3' UTR sequences was inserted downstream of the secreted Gaussia luciferase (GLuc) reporter gene driven by SV40 promoter. A secreted Alkaline Phosphatase (SEAP) reporter driven by a CMV promoter cloned into the same vector served as the internal control for normalization. RASFs were co-transfected with ASK1 luciferase reporter construct (1 μg) with pre-miRNA (100 nM) of miR-17 or negative control pre-miRNA (100 nM) (Life Technologies, CA, USA) using Lipofectamine® 2000 transfection reagent (Life Technologies). After 48 h, the condition media was collected and luciferase activity was assayed using a Secrete-Pair™ Dual Luminescence Assay kit (Genecopia, Rockville, MD). Each experiment was repeated in three independent SF donors and each assay was performed in triplicate.