Fig 6.

STING palmitoylation mediates STING exocytosis in EVs. (A) HEL cells were either uninfected or infected with HSV-1(F) (0.5 PFU/cell) and untreated or treated with H-151 (30 µM) that was added to the culture with the virus. Cell lysates and supernatants were harvested at 48 h post-infection, and EVs were isolated from the supernatants after pelleting at 100,000 × g. Total EVs were analyzed by western blot for STING and CD63. Also, proteins from equal amounts of cell lysates were analyzed for STING, CD63. ICP0 served as a control for infection and β-actin as a loading control. (B) HEL cells were infected with HSV-1(F) (0.5 PFU/cell), and untreated or treated with H-151 (30 µM) that was added to the cultures with the virus. Cells were collected at 3, 24, and 48 h post-infection, and progeny virus production was determined by plaque assays. All values were derived after analyzing samples from three independent experiments. *P  ≤  0.05; **P  ≤  0.01; ***P  ≤  0.001. (C) HEp-2 cells and derivatives expressing either unmodified Flag-tagged STING or Flag-tagged STING mutants were either uninfected or infected with HSV-1(F) (0.5 PFU/cell). Culture supernatants were harvested at 48 h post-infection, and EVs were isolated after pelleting at 100,000 × g. Total EVs were analyzed by western blot for STING. Equal amounts of cell lysates from infected cells were analyzed for STING monomers and dimers and for β-actin.