EBNA3C promotes IRF4-mediated cell proliferation by inhibiting IRF8 induced apoptosis.

<p>A–D) Human Kidney embryonic cells (HEK-293) were transfected with Ctrl-vector and different combinations of expression plasmids for Myc-tagged EBNA3C, Flag-tagged IRF8 and Myc-tagged IRF4. Additionally, cells were transfected with GFP expression vector. Transfected cells were selected for 2 weeks with G418 antibiotic. A) 0.1×10⁶ cells from each set of selected samples were plated and cultured for 6 days. Viable cells were counted at indicated time points by trypan blue dye exclusion technique. (B) G418 selected stable cells were harvested, lysed in RIPA buffer and subjected to immunoblot analyses with indicated antibodies. C–D) HEK-293 cells transfected with different combinations of expression plasmids as indicated in previous experiment and selected similarly as described above with G418 antibiotic. After 2 weeks of selection, GFP fluorescence of each plate was scanned by a PhosphorImager and the area of the colonies was calculated by Image J software. The data represented in bar diagram as the average of three independent experiments. E–J) Lentivirus mediated knock down of IRF4 in EBV negative Ramos cells. Knocked down cells were selected with puromycin to make stable cell lines expressing specific si-RNA against IRF4 and control vector. F) The selected stable cells with GFP fluorescence were monitored by fluorescent microscope. G) 50 million Ramos, stable Sh-Ctrl Ramos, Sh-IRF4 Ramos cells were lysed in RIPA buffer and Western blot was performed to check the expression levels of IRF4. E) Stable Sh-Ctrl Ramos, Sh-IRF4 Ramos, cells were transfected with control vector, Flag-IRF8, Myc-EBNA3C and grown in RPMI medium for 36 hrs. Transfected cells were stained with Propidium iodide and subjected to FACS analysis. H) The bar diagram represents the percentage of apoptosis in each transfected set. The results shown are representative of three independent experiments. I–J) 1×10⁶ stable transfected cells were plated and allowed to grow in RPMI media for 6 days. Viable cells were counted by trypan blue dye exclusion method at indicated time points.