MEF-2 inhibition reduces HTLV-1 LTR transactivation, Tax expression, and viral replication. (A) Transient transfection of Jurkat cells with pU3R-luc (HTLV-1 LTR luciferase reporter construct) as well as plasmids that express Tax, MEF-2A, HDAC9 and MEF-2A shRNA, was done as described in Methods. Before co-transfecting two or more plasmids, each of these plasmids was transfected alone to establish the background levels of luciferase activity. Cells were collected 24 hr post-transfection, lysed and assayed using the dual luciferase assay system. Firefly luciferase activity was normalized with that of Renilla luciferase expressed from phRL/CMV. Each bar represents the average of triplicate samples. Significance among groups was derived by student’s t-test to determine the p-value. (*p < 0.05). (B) MT-2 cells were transfected with either scrambled or shMEF-2 plasmid. Western blot analysis was performed at 24 hr and 48 hr to determine protein levels of MEF-2, Tax, and beta-actin. Data represent one of two separate experiments. (C) To analyze effects of shMEF-2A on virus production, transfected MT-2 cells were washed at 48 hr and incubated in fresh medium for another 24–36 hr. Thereafter, supernatants were assessed for HTLV-1 core protein levels (pg/ml) by the p19-specific ELISA (ZeptoMetrix, Buffalo, NY). (D) MT-2 cells were transfected either with a mock plasmid or MITR/HDAC9 plasmids followed by cell collection at every 24 hr over a 72 hr period. Real-time PCR analyses were performed to determine relative mRNA levels of Tax and p19. Data is representative of at least three independent experiments.