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

Figure 6.

Figure 6

cGAS N-Terminal Domain Determines α-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.