Fig 3.

STING exocytosis is triggered by HSV-1(F) infection but not by a STING ligand and downstream effector binding. (A) HEL cells were uninfected, transfected with 2′3′-cGAMP at a final concentration of 3 µM, or infected with HSV-1(F) at 0.5 PFU/cell. EVs were isolated from the supernatant after pelleting at 100,000 × g and analyzed by western blot for STING or CD63. Also, equal amounts of proteins from total cell lysates were analyzed for CD63 and p-STING. ICP0 served as a control for infection and β-actin served as a loading control. (B) HEL cells and their TBK1 knockdown derivatives 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 centrifugation at 100,000 × g. Samples were analyzed by western blot for STING and CD63. In addition, equal amounts of proteins from total cell lysates were analyzed for STING and CD63. ICP0 served as a control of infection and β-actin as a loading control. (C) HEL cells and the TBK1 KD derivatives were either uninfected or infected with HSV-1(F) or ΔICP0 virus (0.5 PFU/cell). Cell lysates were harvested at 48 h post-infection and analyzed for p-TBK1 (Ser-172) and total TBK1. Probing for β-actin served as a loading control and for VP16 as a control for the infection. (D) HEL cells and the TBK1 KD derivatives were uninfected, infected with HSV-1(F), ΔICP0 virus (0.5 PFU/cell), or transfected with 2′3′-cGAMP (6 µM). The cells were harvested at 48 h post-treatment or post-infection, and equal amounts of proteins from total cell lysates were analyzed for p-STING (Ser-366) and STING. VP16 served as a control for the infection and β-actin as a loading control. Red asterisk marks p-STING (Ser-366).