LMP1-mediated NF-κB activity coordinates a repressive complex to repress miR96 and promote PRMT5 expression. (A) miR96 expression was measured by qRT-PCR in a transformed LCL (60A) after LMP1 knockdown using 2 separate LMP1-specific shRNA preparations. Western blot showing the effect of LMP1 knockdown on LMP1 and PRMT5 expression is also included (bottom panel). LMP1 knockdown resulted in statistically significant increase of miR96 expression at 48 hours with both preparations. (B-C) Two transformed LCLs (60A and SR27) were incubated with the selective inhibitor of IkB kinase α (BAY11, 10 µM) and miR96 expression was evaluated by qRT-PCR. Exposure to BAY11 (10 µM) resulted in a statistically significant increase of miR96 expression at 4 hours in 60A cells and at 2 and 4 hours in SR27 compared with DMSO control. (D-E) The expression of PRMT5 and epigenetic mark, S2Me-H4R3, was evaluated by western blot and confocal microscopy in 60A cells incubated for 4 hours with BAY11 (10 µM). Actin was used as loading control and MCL-1 as control for BAY11 activity. (F-H) One immortalized (D-22) and 1 transformed LCL (60A) were incubated with a selective HDAC1,2 inhibitor (JQ12, 10 µM) (F), a broad-spectrum HDAC inhibitor (AR42, 1 µM) (G) and with a selective HDAC1,3 inhibitor (MS275, 2 µM) (H), or DMSO control. miR96 expression was evaluated by qRT-PCR at the indicated time points. The bar graph shows normalized fold expression of miR96 relative to untreated cells using GAPDH as internal control. *P < .05; **P < .01. Error bars indicate SEM. (I) Western blot of H3K14Ac in D-22 and 60A cells treated for 12 and 24 hours with MS275 (2 µM). Actin was used as loading control.