Bmi-1 expression in EBV-negative HL cells is also NF-κB dependent. (A) Immunohistochemistry for Bmi-1 in primary HL. (Top) Typical nuclear staining of HRS cells (arrow) observed in most patients. Only 3 of 60 patients lacked Bmi-1 expression. (Middle) Typical image showing lack of HRS cell expression of Bmi-1 (arrow). (Bottom) Mantle zone (MZ) B cells and centrocytes (CC) were Bmi-1 positive in control tonsil, whereas centroblasts (CBs) were negative. Images were acquired using a Zeiss Photomicroscope II equipped with a Nikon Coolpix990 camera (Nikon, Kingston upon Thames, United Kingdom) and using Corel Paint Shop Pro v.10 software (Maidenhead, United Kingdom). Top two images were acquired with a 60 ×/1.40 NA oil-immersion objective lens (Nikon, Kingston upon Thames, United Kingdom); the bottom image, with a 40 ×/0.65 NA dry objective lens. (B) Treatment of L428 cells with the NF-κB inhibitor TLCK resulted in the down-regulation of Bmi-1 expression. Changes in Bmi-1 mRNA (top) and protein (bottom). Similar results were obtained with EBV-negative KMH2 cells (data not shown). (C) KM-H2 cells transfected with a plasmid expressing an IκBκ mutant that inhibits NF-κB activation. Expression of the IκBκ mutant resulted in the down-regulation of Bmi-1 expression compared with cells transfected with control vector.