miR-28 regulates proliferation and cell death in lymphoma B cells by dampening the BCR signaling pathway. (A) Extracts of RFP⁺ Ramos BL cells, transduced with control or pre-miR-28–containing retroviral constructs, were immunoblotted with antibodies to phospho-ERK1/2 (T202/Y204), ERK1/2, and phospho-AKT (S473). Numbers beneath the bands show protein quantification after normalization to the α-tubulin loading control signal. Bar graphs on the right show data from 2 independent experiments. (B) AKT phosphorylation was measured by flow cytometry after anti-IgM stimulation of RFP⁺ Ramos cells expressing miR-28 (blue shaded histogram) or scramble RNA (red open histogram). The panel shows representative flow cytometry plots (top) and quantification of 4 independent experiments after normalization to controls (bottom). MFI, mean fluorescence intensity. *P < .05, unpaired Student t test. (C) qRT-PCR of Bcl-2, NFKB2, and IKKB in miR-28 vs control Ramos RFP⁺ BL cells (n = 3). (D) Graphs show miR-28 expression plotted against transcript levels of NFKB2, IKKB, and BCL2 in human primary ABC-DLBCL lymphoma cohorts (data extracted from Iqbal et al²⁶ ). R² and P values are shown. (E) The MD-901 ABC-DLBCL cell line and the Raji and Ramos BL cell lines were transduced with pTRIPZ vectors encoding miR-28 (blue circles) or scramble RNA (red circles). RFP⁺ cells were cultured and counted every day throughout the culture period. Data are from at least 2 independent experiments. *P < .05, unpaired Student t test. (F) Primary splenic B cells were labeled with violet cell tracer, transduced with miR-28 or an empty control retroviral vector, and cultured in vitro with anti-IgM + IL-4. The bottom panel shows representative FACS histograms of GFP⁺ cells 2 days after retroviral transduction (red open line, control; blue shaded histogram, miR-28). The top panel shows quantification of the proportion of cells that have undergone 0 to 5 divisions (n = 2). *P < .05, unpaired Student t test. (G) FACS analysis of cell cycle in RFP⁺ miR-28– or control-transduced Ramos BL cells labeled with propidium iodide and 5-bromo-2′-deoxyuridine (BrdU). Cell-cycle phases and BrdU incorporation in RFP⁺ miR-28– and control-transduced Ramos BL cells are quantified on the right (n = 4). (H-I) 7AAD and annexin V staining (n = 2) (H) and active caspase 3 staining (n = 6) (I) in RFP⁺ miR-28– and control-transduced Ramos BL cells. *P < .05, unpaired Student t test.