Induction of LMP-1 by hIL-10 in P3HR1 BL cells. (A) Total cell extracts of P3HR1 cells incubated with 100 ng/mL hIL-10 for 48 hours were analyzed for LMP-1 expression by SDS-PAGE and immunoblotting. The type III Raji and Jijoye P79 lines were included for comparison. (B) LMP-1 immunofluorescence staining of the IL-10-treated P3HR1 cells (50 ng/mL, 48 hours). The nuclei were stained with Hoechst 33258. (C) LMP-1 and BCL-6 expression of hIL-10-treated Daudi and P3HR1 cells. The cells were cultured in the presence of 100 ng/mL hIL-10 for 2 or 5 days. The membrane was first developed with anti-BCL-6 antibody then stripped and reprobed with LMP-1-specific antibody. The Akata BL/LCL pair was included as controls. (D) Flow cytometric analysis of CD10, CD80, CD83, HLA-II, and ICAM-1 (CD54) antigens in the hIL-10-treated P3HR1 cells. The cells were treated with 100 ng/mL hIL-10 for 48 hours before the staining. The fluorescence intensity in the FL2 channel was measured (logarithmic amplification) and analyzed with the CellQuest Pro program (BD Biosciences, San Jose, CA). On the histogram plots the shaded areas represent the background fluorescence of the secondary Ab-stained cells, whereas the solid black lines denote the specific staining of the untreated and hIL-10-treated cells. The mean fluorescence intensity is indicated. B958-LCL was used as positive staining control.