Fig 1: Functional assays of LSM14A.(A) LSM14A activated ISRE in a dose-dependent manner. The 293T cells (30,000 cells per well in 48-well plates) expressing the ISRE-FLuc and RL-RLuc reporters were transfected with the indicated amounts of GFP (vector) or LSM14A plasmids. The next day, the cells were left uninfected (left panel) or infected with SeV (right panel) for 24h followed by luciferase assay. The FLuc/RLuc ratio was calculated and plotted as fold-increase over the minimum amount of vector plasmid used (6.25 ng). (B) LSM14A cleavage fragments do not activate ISRE. The reporter assays with the indicated LSM14A mutants were performed similarly as in A. The DNA concentrations of 6.25, 12, 25, and 50 ng/well were used. (C) TEVP-mediated cleavage of LSM14A blocks its ability to activate ISRE. The 293T cells containing dox-inducible TEVP and expressing the ISRE-FLuc and RL-RLuc reporters were transfected with 12.5 ng/well, 25 ng/well, or 50 ng/well LSM14A-TEVP plasmid followed by SeV infection. The indicated amount of doxycycline was added to the culture medium 24h prior to DNA transfection and kept for the entire experiment. The reporter assay was performed as described in A. (D) LSM14A requires RIG-I and MAVS for its function. The 293T cells knocked out for the indicated genes were used for the reporter assays as described in A (top panel). 50 ng/well DNA was used. The cells were tested for the expression of the indicated proteins. For detection of RIG-I and MDA5, the cells were exposed to 5 nM IFN-2a alpha for 48h. (E) MAVS does not require LSM14A for its innate immune function. The 293T cell clones expressing or lacking LSM14A were transfected with 12.5 ng/well MAVS and tested for the activation of ISRE as in A. This experiment was performed in the absence of SeV infection. KO-pool was generated by mixing the 293T cell clones that were confirmed for the loss of LSM14A. (F) SeV can activate ISRE in the absence of LSM14A. The 293T cell clones used in E were infected with SeV and the ISRE reporter activity was measured as in A. For all experiments, the data are presented as mean +/- standard deviation for triplicate samples.