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. 2023 Feb 7;97(2):e01975-22. doi: 10.1128/jvi.01975-22

FIG 3.

FIG 3

Sirt1 knockdown promotes HSV-1- or exogenous cytosolic DNA- induced innate immune responses. (A) HEK293T cells were transfected with Myc-Sirt1 and then transfected with control siRNA (SC) or Sirt1-specific siRNA (S1, S2, and S3). At 24 h after transfection, the cells were lysed for immunoblot assays (top). PMA-THP1 cells were transfected with control siRNA (SC) or Sirt1-specific siRNA (S1, S2, and S3). At 24 h after transfection, the cells were subjected to immunoblot assay (bottom). (B and C) PMA-THP1 cells were transfected with control siRNA (SC) or Sirt1-specific siRNA (S1 and S2) for 24 h and then stimulated with HSV-1 (MOI = 1) (B) or HSV60 (1 μg/mL) transfection (C) for 8 h. The cells were lysed for real-time PCR analysis. (D) PMA-THP1 cells were transfected with control siRNA (SC) or Sirt1-specific siRNA (S1) for 24 h and then transfected with HSV60 (1 μg/mL), VACV70 (1 μg/mL), and poly (I:C) (2.5 μg/mL) for 8 h. Then, the cells were lysed for real-time PCR analyses. (E) PMA-THP1 cells were transfected with control siRNA (SC) or Sirt1-specific siRNA (S1 and S2) for 24 h and then infected with HSV-1 (MOI = 1) for 4 h. The cells were lysed for immunoblot assays. (F) PMA-THP1 cells were transfected with control siRNA (SC) or Sirt1-specific siRNA (S1) for 24 h and then infected with HSV-1 (MOI = 1) or VSV (MOI = 1) for 4 h. The cells were lysed for native PAGE or SDS-PAGE assays. (G) PMA-THP1 cells were transfected with control siRNA (SC) or Sirt1-specific siRNA (S1 and S2) for 24 h and then transfected with HSV-60 (1 μg/mL) for 4 h. The cells were lysed for native PAGE or SDS-PAGE assays. (H) PMA-THP1 cells were transfected with control siRNA (SC) or Sirt1-specific siRNA (S1) for 24 h and then transfected with HSV60 (1 μg/mL), VACV70 (1 μg/mL), poly(dA·dT) (dA:dT; 1 μg/mL), and poly (I:C) (I:C; 2.5 μg/mL) for 4 h. The cells were lysed for immunoblot assays. (I) PMA-THP1 cells were transfected with control siRNA (SC) or Sirt1-specific siRNA (S1) for 24 h and then infected with HSV-1 for 24 h. The titers of HSV-1 were determined by a standard plaque assay. β-Actin served as a loading control in all the immunoblot assays. The data are representative of three independent experiments and are presented as means ± SDs. *, P < 0.05; **, P < 0.01; ***, P < 0.001.