Cellular miR-155 is highly induced following EBV infection of resting B cells. (A) Expression of miR-155 at the indicated time points (hours) postinfection with EBV as measured by qRT-PCR. Time zero was immediately following EBV infection. miR-155 levels in 293T and BJAB cells are shown for comparison. This graph presents the kinetics of miR-155 expression during the generation of the newly established LCL line SDLCL. Similar kinetics of miR-155 induction were observed between the primary B-cell state and LCLs by miRNA microarray analysis (data not shown). (B) Absolute quantitation of miR-155 in primary B cells and various EBV-infected B-cell lines. Results shown represent the averages of three independent qRT-PCRs, each performed in duplicate, with standard deviations (SD) indicated.

miR-155 is required for growth of LCLs and DLBCLs but not EBV-positive or EBV-negative BL cells. Growth curves of GFP-positive cells expressing control sponges (black diamonds, GFP; gray triangles, sCXCR4) or a sponge specific for miR-155 (red squares, s155). GFP-positive cells (y axis) are plotted against days postsort (x axis), as determined by FACS. (A) SDLCL. (B) EF3D. (C) IBL-1. (D) BJAB (EBV negative). (E) Raji. (F) Namalwa. Graphs for panels C, D, E, and F are representative of two independent experiments, while the growth curves shown in panels A and B were determined three times. Note that the IBL-1 cells analyzed here were transduced with an MSCV-based retroviral sponge expression vector, while all other cells were transduced with lentiviral sponge expression vectors.

Selective inhibition of miR-155 function in Namalwa cells transduced with an miR-155-specific sponge. Namalwa cells transduced with the control pLCE/GFP lentiviral vector or with the pLCE/s155 sponge vector were sorted for high GFP expression by FACS and then transduced with indicator vectors measuring miR-155 or miR-BHRF1-1 function. miR-BHRF1-1 activity was measured as a control for sponge specificity. 293T cells, which express neither miR-155 (Fig. 1A) nor miR-BHRF1-1, were used as a control. rel., relative.

A miR-155-specific sponge strongly inhibits LCL growth in culture. The LCL SDLCL was transduced with a lentiviral GFP vector expressing a linked miR-155-specific sponge (blue) or a control lentiviral GFP expression vector (red) and allowed to grow for 6 days prior to being sorted by FACS. Levels of GFP-positive cells relative to GFP-negative cells were measured on a FACSCanto instrument immediately postsort (left panel), 6 days postsort (middle panel), and 25 days postsort (right panel). Graphs were generated using FlowJo software and are representative of three independent experiments.

SDLCL cells knocked down for miR-155 function arrest in G₁ and undergo increased apoptosis. (A) Cell cycle analysis of SDLCL cells expressing an miR-155-specific sponge (s155; center panel) or expressing control vectors (GFP and sCXCR4 [left and right panels, respectively]). Results were gated for the GFP-positive population, and this was done to remove sub-G₁ cells as well. Numbers of propidium iodide (PI)-stained cells are plotted on the x axis, and the level of BrdU incorporation is on the y axis. Gates superimposed on each graph measure cells in the G₁ (bottom left), S (top), and G₂ (bottom right) phases of the cell cycle. This FACS plot is representative of three independent experiments that are quantified in panel B. (B) Cell cycle analysis of LCLs (EF3D and SDLCL), as described for panel A, but normalized relative to the GFP control sponge; results are averages from three independent experiments. Error bars represent SD. (C) Annexin V staining of SDLCL cells transduced as described above and gated on GFP-positive cells. The percentages of annexin V-positive cells shown represent averages from three independent experiments. Error bars indicate SD.

Analysis of LCLs expressing a tetracycline-inducible miR-155 sponge. The corresponding experiment was performed as described for Fig. 2, except that the LCLd1 cell line was transduced either with the tetracycline-regulatable GFP expression vector pTRIPZ/GFP or with a derivative (pTRIPZ/s155) bearing the miR-155 sponge in the 3′ untranslated region (UTR) of the gfp gene. After transduction and selection for puromycin resistance, GFP expression was induced by incubation in doxycycline followed by FACS for GFP-positive cells. (A) Growth of GFP-positive LCLd1 cells transduced with either the parental TRIPZ/GFP lentiviral vector or the TRIPZ/s155 derivative. (B) Analysis of miR-155 function in doxycycline-treated LCLd1 cells transduced either with TRIPZ/GFP or with TRIPZ/s155 and then sorted for GFP expression. This experiment was performed as described for Fig. 3 and was normalized to EBV-negative cells. Results of a representative experiment reflective of two independent experiments are shown. (C) LCLd1 cells that had been transduced with TRIPZ/GFP or TRIPZ/s155 were induced with doxycycline, sorted for GFP expression by FACS, and then analyzed for LMP1 or EBNA2 mRNA expression by qRT-PCR. (D) LCLd1 cells were transduced with TRIPZ/GFP or TRIPZ/s155, expanded, and then induced by treatment with doxycycline for the indicated 20-h or 44-h time period. At this point, GFP-positive cells were analyzed for annexin V and 7-AAD expression by FACS.