Fig. 2: A cGAS–STING pathway mediates tissue fluidification-dependent CytoDR genes.
From: Tissue fluidification promotes a cGAS–STING cytosolic DNA response in invasive breast cancer
a, Heatmap of CytoDR DEGs in RAB5A-MCF10.DCIS.com monolayers silenced for the indicated genes (Supplementary Fig. 1a). Data are the ratio of gene expression of each condition relative to mock-scramble-oligos-treated RAB5A-cells. The mean ± s.d. (n = 3 experiments); each-pair two-tailed Student’s t-test are in Source Data Fig. 2a. b, Heatmap of CytoDR DEGs in RAB5A-MCF10.DCIS.com monolayers treated with cGAS inhibitor RU.521(7 μg ml–1), or STING antagonist, H-151 (4 μg ml–1), or TBK1/IKK inhibitor, MRT67307 (20 μM). Data are the ratio of gene expression in each condition relative to vehicle-treated RAB5A-cells. The mean ± s.d. (n = 3 independent experiments), each-pair two-tailed Student’s t-test are in Source Data Fig. 2b. c, Heatmap of CytoDR DEGs in RAB5A-MCF10.DCIS.com monolayers silenced for the indicated genes. The data are the ratio of gene expression in each of conditions relative mock-scramble-oligos-treated RAB5A-cells. The mean ± s.d. (n = 7 experiments), each-pair two-tailed Student’s t-test are in Source Data Fig. 2c. d, Immunoblots of control- (CTR) and RAB5A-MCF10.DCIS.com monolayers with the indicated antibodies (n = 3 independent experiments). e, Still images of cell contours are indicated by pseudo-colouring of EGFP-CDH1, control- (CTR) and RAB5A-MCF10A cells (Supplementary Video 4). Scale bar, 10 μm. f, Scheme depicting cell maximum positive deformation (MPD) and negative deformation (MND). g, Data are the mean ± s.d. of MPD and MND (80 cells per condition in n = 4 independent experiments). Two-tailed Mann–Whitney non-parametric test. h, Consecutive frames (top-left, top-right and bottom-left subpanels) of the same RAB5A-expressing MCF10A nucleus (Supplementary Video 5). Continuous lines with different shades of red represent the profiles obtained via nuclear segmentations. In the bottom-right supanel is reported a superposition of the three profiles shown in the other subpanels. Scale bar, 5 μm. i, Comparison of the nuclear mean square strain (MSS) of control and RAB5A-MCF10A (left panel) or MCF10.DCIS.com (right panel) cell monolayers. The MSS is obtained by tracking and segmenting N nuclei over the time window 4–20 h (N > 5,000 and N > 1,000 for control- and RAB5A-MCF10A monolayers, respectively; >N700 and >N400 for control- and RAB5A-MCF10.DCIS.com monolayers, respectively). Continuous lines are best fitting curves to the data with an exponential model. Insets report the nuclear strain rate as mean ± s.d. (n = 10 randomly populated subsets of cells), two-tailed t-test. j, Levels of LMNA, LMNB1 and RAB5A mRNA in RAB5A- and control-MCF10.DCIS.com monolayers. The data are the mean (n = 7 for LMNA, n = 25 for LMNB1 n = 25 for RAB5A). Two-tailed Mann–Whitney non-parametric test. k, Immunoblots of control- and RAB5A-MCF10.DCIS.com monolayers with the indicated antibodies (n = 3 independent experiments). l. Scatter plot of the expression level of Lamin A/C and Lamin B1 in control and RAB5A-expressing MCF10.DCIS.com monolayers. Data are mean ± s.d. of the integrated density/cell measured in different FOV in n = 3 independent experiments, unpaired two-tailed t-test with Welch’s correction. P values are indicated in each graph.
