Cytokine profiling of miRNA-transduced myeloid cells

Mouse cytokine Th1/Th2 kit from BioRAD (Bio-Plex Pro™ Mouse Cytokine Th1/Th2 Assay #M60-00003J7) and mouse TGF-β1 DuoSet ELISA kit from R&D systems (DY-1679-05) was used to determine the secretory cytokines from Gr-1⁺CD11b⁺ transduced with miR-130a and miR-145 lentivirus vectors or miRNA mimics. These transduced cells were cultured in serum-free medium for 24 h, and supernatants from the cells were collected. The amount of secreted cytokines and the ratio of M1/M2 was calculated by dividing each M1 cytokine (TNFα, IL-12, GM-CSF) to M2 cytokine (TGFβ1, IL-10, IL-4)^{37 (link)}. Briefly, each individual cytokine was measured by Bio-plex, and each individual M1/M2 ratio (TNFα/TGFβ1, TNFα/IL-10, TNFα/IL-4, IL-12/TGFβ1, IL-12/IL-10, IL-12/IL-4, GM-CSF/TGFβ1, GM-CSF/IL-10, and GM-CSF/IL-4) was calculated, and summed up for each of miRNA and control. For preventing the mRNA degradation of TβRII, IGF1R and IRS1 in Gr-1⁺CD11b⁺ cells by miR-130a or miR-145, overexpression constructs without 3′-UTR were utilized including FerH-mouse Tgfbr2 (Protein Expression Laboratory in Frederick National Laboratory for Cancer Research), pReceiver-mouse Igf1r (GeneCopoeia), pBABE-mouse Irs1 (Addgene). The M1 and M2 cytokine ratio was calculated as indicated above.