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. Author manuscript; available in PMC: 2016 Nov 1.
Published in final edited form as: Am J Hematol. 2015 Oct 8;90(11):998–1007. doi: 10.1002/ajh.24136

FIGURE 2. Up-regulation of miR-181a effectively controls Th1 responses in vitro.

FIGURE 2

(A) Intracellular staining of IFN-γ, IL-4, IL-17 and Foxp3 in CD4+ T cells infected with LV-Ctrl, LV-181a, and LV-181a+IFN-γ. Mock infected CD4+ T cells served as negative control. Cells were examined by intracellular staining 3 hours after stimulation with PMA and ionomycin after fixation by brefeldin A. Data are representive of three independent experiments. (B) ELISA analysis of cytokines (IFN-γ, IL-4 and IL-17) in culture media of CD4+ T cells according to the protocol described in Materials and Methods. Levels of IFN-γ and IL-17 were significantly decreased while IL-4 was significantly increased. (C) Analysis of proliferation (MTS) and apoptosis (Annexin V and 7-AAD) of CD4+ T cells 3 hours post-stimulation with PMA, ionomycin and fixation with brefeldin A. In cells overexpressing miR-181a, the OD value measured at 490nm of CD4+ T cells was significantly lower while Annexin V and 7-AAD positive cells was higher; these changes returned to normal when IFN-γ was re-expressed. (D) Representative H&E straining of human CD4+ T cells activated by PHA for 24 and 48 hours. Final magnification 100× (Olympus, DP-2-BSW). ** p < 0.01, *** p < 0.001. All values represent mean±SD. Data are from three independent experiments.