Figure 2. miR-21 acts as a potent negative regulator of IL-10-producing B cells.
(A) miR-21 is aligned with the highly conserved 3’UTR of IL-10 mRNA. (B, C, E) CD19+ B cells were purified by MACS from naive B6 mice, cultured for 24 h under LPS/PMA/ionomycin/monensin conditions, and transfected by nucleofection with miR-21 inhibitor, mimic or negative-control oligonucleotides. (B) miR-21 expression was analyzed by qRT-PCR using U6 snRNA as an endogenous control. (C, E) IL-10 levels were measured by intracellular staining and flow cytometric analysis. The numbers represent the frequencies of CD19+ B cells. (D, F) CD19+ B cells were cultured as described in B, C, and E, except monensin was not added. The culture supernatants were collected after 24 h and used for the estimation of IL-10 secretion by ELISA. The data are representative of 3 independent experiments. *P < 0.05 and **P < 0.01.