Fig. 3: Nuclear envelope ruptures activate cGAS in fluidized monolayers.
From: Tissue fluidification promotes a cGAS–STING cytosolic DNA response in invasive breast cancer
a, Immunofluorescence images of doxycycline-treated, EGFP-cGAS control (CTR) and RAB5A-expressing (RAB5A) MCF10.DCIS.com cells (n = 4 experiments) seeded either sparsely or as confluent monolayers. Scale bar, 10 μm. Red arrow point to EGFP-cGAS perinuclear foci b, Scatter plot of the percentage of MCF10.DCIS.com cells with perinuclear cGAS enrichment is expressed as the mean ± s.d. (>15 FOV per experimental condition in n = 4 independent experiments), unpaired two-tailed t-test with Welch’s correction. c, Quantification of cGAMP levels by ELISA from control (CTR) and RAB5A-expressing (RAB5A) MCF10.DCIS.com cell extracts. Data are the cGAMP amounts (ng) per mg of total cell extract expressed as the mean ± s.d. (n = 8 experiments), two-tailed Mann–Whitney non-parametric test. d, Snapshot of time-lapse (Supplementary Video 6) EGFP-3NLS-expressing control (CTR) and RAB5A MCF10.DCIS.com monolayers (n = 2 independent experiments), displaying events of NE rupture and EGFP-3NLS leakage (red arrowheads). Scale bar, 10 μm. e, Scatter plot of the number of nuclear envelope rupture events per FOV reported as mean ± s.d. (10 FOV per experimental conditions in n = 2 independent experiments), two-tailed Mann–Whitney non-parametric test. f, CLEM analysis of cGAS perinuclear foci. RAB5A-expressing MCF10.DCIS.com monolayers transfected with EGFP-cGAS were plated on MaTek dishes with grids. Cells identified on grids by confocal microscopy were processed for electron microscopy and z-axis serial sections were stained with gold-labelled anti-GFP antibody to detect EGFP-cGAS (right). Dashed boxes indicate regions that were progressively magnified in EM. The bottom images show a 3D tomographic reconstruction (left) and 3D models of an NE rupture site (right panels). Arrowheads indicate the site of NE rupture (n = 2 independent experiments).
