Fig 2: cGAS N-Terminal Domain Determines a-Satellites’ Association, Cytosolic Retention, and Activation in the Nucleus(A) (Left) Schematics of cGAS deletions and (right) confocal microscopy of DCs transduced with human full-length catalytically inactive cGAS, the N-terminal part of cGAS (cGAS 1–212), or the C-terminal part (cGAS 161–522) fused to GFP in pTRIP-SFFV. GFP channel is shown in black on white. One representative donor of n = 4 donors in 2 independent experiments. Scale bars, 10 µm.(B) (Left) Schematics of cGAS deletions and (right) confocal microscopy of DCs transduced with human full-length catalytically inactive cGAS or cGAS 1–160 in pTRIP-SFFV. GFP channel is shown in black on white. One representative donor of n = 4 donors in 2 independent experiments. Scale bars, 10 µm.(C) GFP, CD86, and SIGLEC1 expression in DCs after transduction with a GFP-NLS, GFP-NLS-cGAS, GFP-cGAS 161–522, or GFP-NLS-cGAS 161–522 lentivector in pTRIP-SFFV in the presence or absence of Vpx. One representative donor of n = 6 donors in 3 independent experiments.(D) CD86 and SIGLEC1 expression in DCs transduced as in (D); n = 6 donors in 3 independent experiments. One-way ANOVA with post hoc Tukey test.(E) Confocal microscopy of Cgas-/- mouse bone marrow-derived DCs transduced with GFP-NLS, GFP-cGAS, GFP-NLS-cGAS, GFP-cGAS 1–160, or GFP-cGAS 161–522 in pTRIP-SFFV lentivectors. GFP channel is shown in black on white. One representative mouse of n = 2. Scale bars, 10 µm.(F) Expression of Ifit1 in Cgas-/- mouse bone marrow-derived DCs transduced with GFP, GFP-NLS, GFP-cGAS, GFP-NLS-cGAS, GFP-cGAS 1–160, or GFP-cGAS 161–522 in pTRIP-SFFV lentivectors, untreated or treated with reverse transcriptase inhibitors (AZT + NVP) or transfected with cGAMP; n = 4 mice combined from 2 independent experiments. Bars represent geometric means. One-way ANOVA with Sidak test on log-transformed data.(G) Quantification of GFP intensity in CENP-B foci in the nucleus, normalized over mean nuclear GFP intensity, in DCs transduced with GFP-NLS or the indicated GFP-cGAS lentivectors, in pTRIP-SFFV; n = 140 foci per construct were quantified in 7 or 8 independent cells per construct. Each dot represents a single CENP-B focus. Means and SDs are represented. One representative donor of n = 4 donors in 2 independent experiments. One-way ANOVA with post hoc Tukey test.***p < 0.001, ****p < 0.0001, ns, non-significant. See also Figure S5.

Fig 3: 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).