Inhibition of miR-24 in U2932 EBNA2-transfected cells induces ICOSL 3′ UTR luciferase activity. (A) Assessment of miR-24 levels by RT-qPCR in U2932 MPA vector and EBNA2 cells at 48 hours after control inhibitor and anti–miR-24 transfection. RNU6 housekeeping gene was used to normalize miR-24 fold change. (B) ddPCR shows the copy number of miR-24 per μL in each transfection. RNU6 housekeeping gene was used to normalize miR-24 copy number per μL in each sample. (C) Luciferase (Luc) assay was performed at 48 hours after transfection with control (cntr) inhibitors and anti–miR-24 in triplicates for each sample. Increased activity of ICOSL 3′ UTR is observed after inhibition of miR-24 in U2932 EBNA2 cells. Luc activity was measured as relative luminometer units (RLU) ratio between ICOSL 3′ UTR and the control 3′ UTR, to normalize RLU for each sample. The histogram demonstrates fold change in luc activity compared with U2932 MPA vector transfected with the cntr inhibitor. All experiments were repeated at least 3 times. One-way ANOVA and Tukey multiple comparisons test was applied to evaluate statistical significance; ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.