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. 2011 Jan 5;85(6):2666–2685. doi: 10.1128/JVI.01532-10

FIG. 2.

FIG. 2.

Immunofluorescence analysis showing a block in nuclear translocation of angiogenin by neomycin treatment and its effect on KSHV+, KSHV−, EBV+, and EBV− B-lymphoma cell, PEL cell, and LCL survival. (A) BCBL-1 cells were treated with the indicated concentrations of neomycin (Neo) for 3 days, fixed, permeabilized, stained for angiogenin (green), and counterstained with DAPI (blue). Arrows indicate angiogenin nuclear staining in BCBL-1 cells. Magnification, ×40. The enlarged images show a single cell displaying nuclear localization of angiogenin. Magnification, ×80. (B) Nuclear and cytoplasmic extracts were prepared from BCBL-1 cells either untreated (lane 1) or treated with 10 μM neomycin (lane 2), 200 μM neomycin (lane 3), and 100 μM paromomycin (Paro; lane 4) and Western blotted with antiangiogenin antibody (top two panels). The nuclear extract blot was stripped and probed with anti-lamin B and antitubulin antibodies (bottom two panels). (C) Supernatants harvested from BCBL-1 cells treated with the indicated concentrations of neomycin were used to measure the levels of angiogenin secretion by ang-ELISA. (D, E, and F) Cell survival assay upon neomycin treatment. The MTT assay was done at 3 days posttreatment for the indicated cell lines with increasing concentrations of neomycin. **, P < 0.001; ***, P < 0.0001.