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. 2019 Feb 26;26(9):2377–2393.e13. doi: 10.1016/j.celrep.2019.01.105

Figure 4.

Figure 4

Confinement-Induced Nuclear Envelope Rupture Does Not Activate the cGAS-STING-IRF3 Axis

(A) Scheme of the cell confiner.

(B) Immunoblot of cGAS, STING, and vinculin in HeLa cells and HeLa cells transduced with mCherry-cGAS E225A/D227A, BFP-2A-STING, and GFP-IRF3 (HeLa STING).

(C) Sequential images of HeLa STING transfected with 4 μg/mL HT-DNA. Transfection was performed at time = 0 min. Binding of mCherry-cGAS E225A/D227A to the transfected DNA is shown at time = 100 min and accumulates over time. Formation of GFP-IRF3 foci and vesicles in the cytoplasm are shown at time = 150 min. GFP-IRF3 translocation peaked at time = 165 min. One representative cell for n = 2 independent experiments. Scale bars, 10 μm.

(D) Quantification of cells showing GFP-IRF3 nuclear translocation after confinement, confinement and transfection with HT-DNA, or only transfection with HT-DNA. Cycling HeLa cells, which express endogenous cGAS, were stably transduced as in (B) One-way ANOVA with post hoc Tukey test; ns, not significant; ∗∗∗∗p < 0.0001; data pooled from 3 independent experiments.

(E) Sequential images of HeLa cells stably transduced as in (B) immediately after confinement at 3 μm (time = 0 min; left) and after 6 h, 45 min from confinement (time = 345 min; right). Arrows indicate cells with NE ruptures as shown by bright mCherry-cGAS E225A/D227A foci in the nucleus. One representative field. Scale bars, 10 μm.

(F) Sequential images of HeLa cells stably transduced as in (B) subjected to 3 μm confinement and transfected with 4 μg/mL HT-DNA immediately after confinement at 3 μm (time = 0 min; left) and after 6 h, 45 min from confinement (time = 345 min; right). Arrows indicate cells with mCherry-cGAS spots in the cytoplasm and with consequent translocation of GFP-IRF3 in the nucleus. One representative field. Scale bars, 10 μm.

(G) Sequential images of HeLa STING cells transfected with 4 μg/mL HT-DNA, after transfection (time = 0 min), and 345 minutes later. For quantification in (D), only cells with bright GFP-IRF3 foci in the cytoplasm were quantified to exclude cells in which GFP-IRF3 translocate due to cGAMP transfer via gap junctions. Scale bars, 10 μm.

(H) Expression of GFP-NLS, GFP-FLAG-cGAS E225A/D227A (∗), GFP-NLS-FLAG-cGAS E225A/D227A (∗∗), endogenous cGAS (ˆ), and actin in DCs transduced with the corresponding lentivectors (representative of 2 independent donors).

(I) Expression of BFP, CD86, and SIGLEC1 in DCs as in (H) 48 h after infection with BFP-reporter HIV-1 and HIV-2 viruses and 24 h after transfection with HT-DNA or cGAMP (n = 2 independent donors).