Extended Data Fig. 3: SIRT1 inhibition abolishes IRF3 LLPS and IFN signaling.
a, Immunofluorescence microscopy and DAPI staining of IRF3–GFP in IRF3–GFP stable cells pre−treated with DMSO or EX527 (20 μM), followed by infection for 8 h with HSV-1 (left). Scale bar, 5 μm. Quantified average number of nuclear IRF3 puncta and the percentages of cells with nuclear IRF3 puncta were shown (right). b, HeLa cells infected for 12 h with HSV-1 were grown on collagen-coated microchamber slides. After fixation, in situ PLA for IRF3/IRF7 was performed with α-IRF3 and α-IRF7 antibodies. The PLA-detected proximity (PROX) complexes are represented by the fluorescent rolling circle products (red dots) (left). Scale bar, 5 μm. Quantification of the PROX score is shown as means ± SD (right). c, IFN-β-Luc, PRD I-III-Luc and IFN-α-Luc activity in HEK293T cells pre-treated with DMSO or EX527 (20 μM) and infected for 12 h with SeV. d, qPCR analysis of Ifnb1 and Ifna mRNA level in RAW264.7 macrophages pre-treated with DMSO or EX527 (20 μM) and infected for 12 h with VSV (MOI, 0.1) or HSV-1 (MOI, 10). e, Immunoblot analysis of SIRT1 knockdown efficiency with independent sh-SIRT1 (#1 to #4 independent constructs) in HEK293T cells. f, IFN-β-Luc, PRD I-III-Luc IFN-α-Luc activity in HEK293T cells depleted for SIRT1 with sh-SIRT1 #1 and stimulated for 12 h with SeV. g, qPCR analysis of sh-SIRT1 #1 & #2 efficiency (left panel), IFNB1 and IFNA mRNA level (middle and right) in control and SIRT1-depleted HEK293T cells followed by SeV infection at the indicated time points. h, qPCR analysis of Sirt1 mRNA (left), Ifnb1(middle) and Ifna (right) mRNA in RAW264.7 cells transfected with siRNA (Co.) or si-Sirt1, followed by infection for various times (horizontal axis) with SeV; results are represented relative to those of the control gene Gapdh. Data are representative of three independent experiments (a, b). n = 6 (a, b) or 3 (c, d, f–h) biological independent samples. Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (a (right), b (right), c, d, f–h).
