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. 2020 Mar 18;16(3):e1008335. doi: 10.1371/journal.ppat.1008335

Fig 5. STING recruits NLRP3 to the ER and promotes the inflammasome formation.

Fig 5

(A–D) Hela cells were co-transfected with pFlag-STING, TM5-deleted STING(delTM5) and pHA-NLRP3 (A), transfected with pFlag-NLRP3 and infected with HSV-1 (MOI = 1) for 4 h or transfected with HSV120 (3 μg/ml) for 4 h (B, C and D). Sub-cellular localization of HA-NLRP3 (green) (A and D), Flag-STING (red) or TM5-deleted STING(delTM5) (green) (A), ER Blue (blue) (A, D), Flag-NLRP3 (red) (B, C), Calnexin (green) (B), DAPI (blue) (B and C), and Calnexin (green) (C) were examined by confocal microscopy. (E) TPA-differentiated THP-1 macrophages were infected with mock or HSV-1 (MOI = 1) for 4 h. Sub-cellular localization of NLRP3 (green), Calnexin (red) and DAPI (blue) were examined by confocal microscopy. (F–H) Hela cells were co-transfected with pFlag-STING and pHA-NLRP3 (F). THP-1 macrophages were infected with HSV-1 (MOI = 1) for 4 h (G). Primary MEFs were primed with LPS (1 μg/ml) for 6 h and infected with HSV-1 (MOI = 1) for 4 h (H). HA-NLRP3, Flag-STING, STING, ERGIC p58, GM130 (cis Golgi), Calnexin (ER), IFI16, AIM2 and GAPDH in whole cell lysate (WCL) and purified ER were determined by Western-blot analyses. (I, J) Hela cells stably expressing sh-STNG were transfected with pFlag-NLRP3 and infected with HSV-1 (MOI = 1) for 4 h or transfected with HSV120 (3 μg/ml) for 4 h. Sub-cellular localization of Flag-NLRP3 (red) (I), Calnexin (green) (I), DAPI (blue) (I), HA-NLRP3 (red) (J) and ER blue (J) were examined by confocal microscopy. (K–M) Hela cells stably expressing sh-STING were transfected with pHA-NLRP3 and infected with HSV-1 (MOI = 1) for 4 h (K). THP-1 cells stably expressing sh-STING were differentiated to macrophages, and infected with HSV-1 (MOI = 1) for 4 h (L). Primary MEFs stably expressing sh-STING were primed with LPS (1 μg/ml) for 6 h, and infected with HSV-1 (MOI = 1) for 4 h (M). HA-NLRP3 (K), NLRP3 (L, M), STING (K–M), Calnexin (ER) (K–M) and GAPDH (K–M) in WCL and purified ER fraction were determined by Western-blot analyses. Densitometry of the blots were measured by Image J.