Profiling of immune related genes silenced in EBV-positive gastric carcinoma identified novel restriction factors of human gammaherpesviruses
Fig 7
IRAK2 and MAL are two IRGs suppressing lytic replication of EBV and KSHV.
(A, B) Protein expression of V5-tagged IRAK2 (A) or MAL (B) in stably transduced AGS-BX cells was verified by immunoblotting. (C) Newly produced EBV viruses from TPA/NaB treated or untreated AGS-BX cells stably expressing V5-IRAK2, V5-MAL, or the vector control, were titrated using Raji cells via flow cytometry. (D) Representative diagram of flow cytometry for (C). AGS cells were used to determine the background. (E) Protein expression of V5-tagged IRAK2 or MAL in transiently transfected HEK293.r219 cells was verified by immunoblotting. Protein expression of KSHV LANA was also probed. (F) Expression of GFP and RFP proteins in TPA/NaB treated or untreated HEK293.r219 cells transiently transfected with V5-IRAK2, V5-MAL, or the vector control, was visualized by fluorescence microscopy (scale bar = 1mm). (G) KSHV genome copies (ORF73/LANA DNAs) in TPA/NaB treated HEK293.r219 cells transiently transfected with V5-IRAK2, V5-MAL, or the vector control, were measured by qPCR assays at 24 hpi. (H) Expression of KSHV lytic genes (ORF50/Rta, K8/K-bZIP, and ORF26) in TPA/NaB treated or untreated HEK293.r219 cells transiently transfected with V5-IRAK2, V5-MAL, or the vector control, were measured by RT-qPCR assays at 24hpi. All of above results are based on n = 3 independent repeats and presented as mean ± SEM (* p<0.05; ** p<0.01; *** p<0.001, two-tailed paired Student t-test).
