Extended Data Fig. 6: Fluidized MCF10.DCIS.com monolayers display an increase nuclear size and enrichment in PRC2-targets.
From: Tissue fluidification promotes a cGAS–STING cytosolic DNA response in invasive breast cancer
a. Immunofluorescence images of nuclei in control (CTR) and RAB5A-(RAB5A)-MCF10.DCIS.com monolayers (n = 3 experiments). Scale bar 20 μm. b. Scatter Plot of the nuclear area per field of view is the mean ± s.d. (> 100 FOV/experimental conditions in n = 3 independent experiments), two-tailed Mann–Whitney non-parametric test. c. Transcription factor enrichment analysis of DEG in MCF10.DCIS.com over control monolayers and entries of the ChEA and ENCODE databases. In the Bars are one-sided P-value (combination of Fisher’s exact test and deviation from expected rank for random input gene-set). Bars are coloured according to the transcription factor/target-gene interactions from ChIP-seq/chip experiments database where they have been found enriched. d. GSEA Enrichment plot of the PRC2 targets gene set in the CGP collection of the GSEA Molecular Signatures Database using DEG in RAB5A-MCF10.DCIS.com monolayers. The green curve is the ES (enrichment score) curve, which is the running sum of the weighted enrichment score obtained from GSEA software, while the normalized enrichment score (NES) and the corresponding one-sided test is shown. e, f. Box plot of the mRNA expression levels of EZH2 and SUZ12 in RAB5A-MCF10.DCIS.com monolayer over control cells silenced with the indicated oligos. Data are the mean ± s.d. (n = 3 independent experiments). Values were normalized to the controls of each experiment. g. Visual representation of genomic tracks for ChIP-seq signal enrichment and SAMMY-seq fractions comparison for a representative chromosome (chr 16). For each position along the chromosome (x axis) the enrichment signal (y axis) is the normalized log2 ratio of ChIP over input control samples sequencing reads for ChIP-seq and the normalized log2 ratio of S4 over S3 sequencing (S4/S3) for SAMMY-seq. The data shown in the genomic tracks (from top to bottom) are: three SAMMY-seq replicates on CTR and RAB5A samples; two H3K9me3 ChIP-seq replicates (performed with two alternative antibodies - see methods) on CTR and RAB5A. h. Average SAMMY-seq enrichment signal over heterochromatic regions (metaprofile). SAMMY-seq experiment profiles computed on CTR and on RAB5A-expressing samples are reported (3 replicates for each condition depicted with lines of different colors - see colour legend). The heterochromatic regions were selected based on H3K9me3 ChIP-seq (with ab-176916-Abcam antibody) enriched domains (n = 78 domains). The x-axis reports the relative position with respect to the start and end borders of the considered domains (domain bodies rescaled) in addition to 1Mb flanking regions with absolute (unscaled) coordinates. The y-axis reports the average SAMMY-seq enrichment across all the considered domains. Similar results are obtained by considering either the S4/S2 or the S4/S3 SAMMY-seq fractions comparisons (log2 ratios) to highlight the location of the less accessible and soluble chromatin fraction. i. mRNA expression levels of IFI27, IFI44, IFI44L, IFI6, OASL and RAB5A in RAB5A- MCF10.DCIS.com cells over control cells injected into mammary fat pads of mice. After one week, mice were fed with doxycycline to induce RAB5A expression and the primary tumours were isolated 4 weeks after doxycycline treatment. Data are the mean (n = 2 independent experiments). Values were normalized to the controls of each experiment. P values are indicated in each graph.
