dataesr/scientific-paragraphs-categorization huggingface dataset:
e. mRNA expression levels of the indicated genes in control-and RAB5A-cells seeded at jamming density (fluidized monolayer RAB5A). The same cells were subsequently replated sparsely in the continuous presence of doxycycline to ensure the maintenance of the transgene expression before extracting RNA. Data are the mean ± s. d. (n = 3 independent experiments). Values were normalized to the controls of each experiment, unpaired two-tailed t-test with Welch's correction. f. mRNA expression levels of IFI6, IFIT1, ISG15 and OASL in MCF10. DCIS. com monolayers seeded at jamming density incubated or not with hypotonic medium (composed of a 1:1 mix of media and H 2 O + 1 mM CaCl 2 + 1 mM MgCl 2 ) at the indicated time points. Data are expressed as mean (n = 3 independent experiments). Values were normalized to the controls of each experiment.
All values are significantly different with a p value less than 0. 05, each-pair two tailed Student's t-test. g. Migration parameters, obtained from PIV, of control (CTR) or RAB5A-(RAB5A)-MCF10. DCIS. com monolayers treated with hypotonic media for 48 h (Supplementary Video 2), as in 2f. Left panel: migration speed v m. Central panel: spatial velocity correlation function C vv evaluated over the time window 8-40 h. In the inset are reported the corresponding correlation lengths L c obtained from stretched exponential fits on C vv curves (black dashed lines in the main panel). Right panel: root mean square velocities in the center of mass reference frame. Velocity fields are computed over 4 FOVs per condition (each FOV including more than 2 × 10 3 cells). Data are averages over different FOVs (right and left panel) and different time intervals (central panel).
Shadowed error bars in left and right panel are evaluated as the standard deviation over the different FOVs. Error bars in central panel are the standard deviation of the mean over 5 different time intervals. h. mRNA expression levels of IFI27, IFI44, IFI44L, IFI6, IFIT1, IFIT3, ISG15, OASL and RAB5A in MCF10. DCIS. com monolayers subjected to different conditions (doxycycline-induction of RAB5A-expression, hypotonic treatment or the combination of both) over control cells. Data are the mean (n = 2 independent experiments). Values were normalized to the controls of each experiment. https://doi. org/10. 1038/s41563-022-01431-x Extended Data Fig. 3 | Tissue fluidification-dependent CytoDR is mediated by RAB5A in HaCat. a-c. Migration parameters of HaCat monolayers, obtained from nuclear tracking.
acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi. org/10. 1038/s41563-022-01431-x. MethodsA list of antibodies and dilutions is in Supplementary Table1. Reagents, oligos and QRT-PCR assays are detailed in Supplementary Table2. A list of organoids with relevant information is in Supplementary Table3. Supplementary Methods are in Supplementary Information.
63clear elasticity (Young's modulus) using atomic force microscopy (AFM)-based force indentation of the nuclear surface through the cell cortex of control and RAB5A-MCF10. DCIS. commonolayersseeded at jamming density. Data are the mean ± s. e. m. (100 cells/condition in n = 3 independent experiments), two-tailed Mann-Whitney non-parametric test. Scale bar, 200 μm. b,c. Quantification of cytoplasmic rheology using genetically encoded multimeric nanoparticle (GEM)37. b)Time-and ensemble-averaged Mean Square Displacement (MSD) plots for control (blue line) and RAB5A (red line) in MCF10. DCIS. commonolayersseeded at jamming density. c)Cumulative distribution function showing the coefficient of diffusion, CDF, (D eff ) data of for control (blue line) and RAB5A (red line) MCF10. DCIS. commonolayersseeded at jamming density.
The rates of nuclear AR variation were extrapolated through a robust fit in R using the 'robustbase' package http://robustbase. r-forge. r-project. org/, where the rate of AR changes as function of time is the slope of the fitted curve. Scatter plot data of mean ± S. D. (114 cells/control and 61 cells/RAB5A of n = 3 independent experiments) of the rate of A/R changes, two-tailed Mann-Whitney non-parametric test. f. Images of the SUN2-stained NE of control and RAB5A-MCF10. DCIS. com cells (n = 3 experiments), seeded at jamming density. Scale bar 2 μm. g. Scatter plot of EOP quantifications expressed as mean ± s. d. (> 181 cells/ condition in n = 3 independent experiments), two-tailed Mann-Whitney nonparametric test. h. Quantification of NE fluctuations in EGFP-Nesprin-expressing, control and RAB5A-MCF10.
Immunofluorescence images of nuclei in control (CTR) and RAB5A-(RAB5A)-MCF10. DCIS. commonolayers (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. comover 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. commonolayers. 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 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. comcells 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. https://doi. org/10. 1038/s41563-022-01431-x.
a. mRNA expression levels of CDH1, AXIN2, CDH2, EGF, MMP13, and ZEB1 in RAB5A-MCF10. DCIS. commonolayersovercontrolcells. Data are the mean ± s. d. (each dot represents an independent experiment). Values were normalized to the controls of each experiment. b. Phase-contrast images of control empty vector (CTR) and RAB5A-expressing (RAB5A) MCF10. DCIS. commonolayers(n= 5 independent experiments). Scale bar, 20 μm. c. Immunofluorescence images of doxycycline-treated control (CTR) and RAB5A-(RAB5A) MCF10. DCIS. comspheroids(arepresentativeimagefrom 5 independent experiments) stained as indicated. Scale bar 20 μm. d. Heatmap representing color-coded expression levels of differentially expressed EMT genes in RAB5A-MCF10. DCIS. commonolayerssilencedforYAPandTAZ. The data are the ratio between the level of gene expression in each of the conditions tested relative to those of mock-treated RAB5A-cells.
The mean ± s. d. (at least n = 3 independent experiments), two tailed Student's t-test (siRNA versus RAB5A-expressing cells) are reported in Source data_Figure 9D. e. mRNA expression levels of YAP and TAZ in RAB5A-MCF10. DCIS. comovercontrolcellssilencedwith the indicated oligos. Data are expressed as mean ± s. d. (n = 3 independent experiments). Values were normalized to the controls of each experiment. f. Heatmap representing color-coded expression levels of differentially expressed EMT genes in RAB5A-expressing MCF10. DCIS. commonolayerssilencedfor the indicated genes. This analysis was carried out on the same total mRNA from the experiment shown in Fig. 2a. The effectiveness of silencing is reported in Supplementary Fig. 4a. The mean ± s. d. (at least n = 3 independent experiments), two tailed Student's t-test (siRNA versus RAB5A-expressing cells) are reported in Source data_Figure 9F.
g. Heatmap representing color-coded expression levels of differentially expressed EMT genes in RAB5A-MCF10. DCIS. commonolayerstreatedwith the following small molecules: cGAS inhibitor, 7 μg/ ml RU. 521, or the STING antagonist, 4 μg/ ml H-151. This analysis was carried out on the same total mRNA from the experiment shown in Fig. 2b. The data are the ratio between the level of gene expression in each of the conditions tested relative to vehicle-treated RAB5A-expressing cells. The mean ± s. d. (at least n = 3 independent experiments) with two tailed Student's t-test are reported in Source data_Figure 9G. h. Scatter plot of the speed of wound closure of control (CTR) and RAB5A-expressing MCF10. DCIS. commonolayerstreatedwithvehicleor the cGAS inhibitor, 7 μg/ ml RU. 521, or the STING antagonist, 4 μg/ ml H-151 (SupplementaryVideo 11).
Data are the mean ± s. d. (n = 3 independent experiments, in which 5 FOV/condition/experiment were analysed), two-tailed Mann-Whitney non-parametric test. i. Scatter plot of control (CTR) and RAB5A-expressing MCF10. DCIS. commonolayersinvadinginto a Matrigel coated wound and treated with vehicle or the cGAS inhibitor, 7 μg/ ml RU. 521, or the STING antagonist, 4 μg/ ml H-151 (SupplementaryVideo 12). Data are the mean ± s. d. (n = 3 independent experiments, in which 5 FOV/condition/ experiment were analysed), two-tailed Mann-Whitney non-parametric test, is indicated in the figure. j. Heatmap representing color-coded expression levels of differentially expressed CytoDR genes in RAB5A-MCF10. DCIS. commonolayersshown in Supplementary Fig. 10ftreated with the indicated small molecules: cGAS inhibitor 7 μg/ ml RU.
Acknowledgements We thank  M. Riboni ,  S. Minardi ,  V. Dall'Olio  and  L. Tizzoni  at  Cogentech  for gene expression profiling and qRT-PCR services;  D. Parazzoli ,  S. Barozzi  and the imaging unit of IFOM cell culture facility for technical assistance;  F. Pramotton ,  A. Poli  and the cleanroom facilities of the  Binning and Rohrer Nanotechnology Center  for assistance with the microchannel apparatus. Thank you to the  Biobankl for Translational and Digital Medicine Unit  ( B4MED ) of the  European Institute of Oncology  for support in providing breast cancer samples. This work was supported by  ERC-Synergy  ( 101071470-SHAPINCELLFATE ) to G. S. ;  Associazione Italiana per la Ricerca sul Cancro  ( AIRC IG#18621  and 5Xmille# 22759  to G. S. , M. F. and C. T. ; AIRC-IG-# 21416  to M.
F. ; MyFirstAIRCgrant MFAG# 22083  to F. G. and S. V. ; AIRC-IG # 21762  and  AIRC 5×1000#21091  to F. d. F); the  Italian Ministry of University and Scientific Research  ( PRIN 2017 ,  2017HWTP2K  to G. S. ;  PRIN-2015SJLMB9  to M. F. and F. d. F. ); the  European Research Council  ( ERC  advanced grant# 835103  to F. d. F. );  Progetto AriSLA 2021 'DDR & ALS; FRRB-Fondazione Regionale per la Ricerca Biomedicaunder the frame of EJP RD , the  European Joint Programme on Rare Diseases  with funding from the  European Union 's  Horizon 2020 research and innovation programme  under the  EJP RD COFUND-EJP N° 825575  to F. g. F. ; from  LABEX DCBIOL  ( ANR-10-IDEX-0001-02 PSL*  and  ANR-11-LABX-0043 ),  ANR   ANR-17-CE15-0025-01  and  ANR-18-CE92-0022-01 ,  INSERM 19CS007-00 ,  INCA PLBIO  to N.
Data availability RNA-seq data of MCF10A, MCF10. DCIS. com cells and organoids are deposited in the Gene Expression Omnibus (GEO) and European Genomephenome Archive (EGA), with the respective accession numbers: GSE183479 RNA-seq, GSE183539 SAMMY-seq, GSE183407 ChIP-seq, GSE205108 RNA-seq of organoids. Other data generated or analysed during this study are included in the Supplementary Information and are available from the corresponding authors upon request. Source data are provided with this paper.
Code availability All custom code and scripts used in this study are available on request from the corresponding authors or online: Matlab for Nuclei dynamics and mechanics (https://doi. org/10. 5281/zenodo. 7117355); Wound Healing analysis tool (https://doi. org/10. 5281/zenodo. 7124712); plugins Fiji for micronuclei, yH2AX foci, H3K27me3 (https://doi. org/10. 5281/ zenodo. 7115350).
1 )Fig. 2 | 12 Fig. 2 | A cGAS-STING pathway mediates tissue fluidification-dependent CytoDR genes. 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 mockscramble-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).
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 controland 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.
acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi. org/10. 1038/s41563-022-01431-x. MethodsA list of antibodies and dilutions is in Supplementary Table1. Reagents, oligos and QRT-PCR assays are detailed in Supplementary Table2. A list of organoids with relevant information is in Supplementary Table3. Supplementary Methods are in Supplementary Information. https://doi. org/10. 1038/s41563-022-01431-x https://doi. org/10. 1038/s41563-022-01431-x P < 0. 0001 P < 0. 0001 Human early breast cancer lesion P = 0. 0002 ( l o w ) 5 A cGAS-positive cells (%) P < 0. 0001 ns P = 0. 0002.
Extended Data Fig. 5 | Mechanical characterization of nuclei in control and fluidized tissues. a. 63clear elasticity (Young's modulus) using atomic force microscopy (AFM)-based force indentation of the nuclear surface through the cell cortex of control and RAB5A-MCF10. DCIS. commonolayersseeded at jamming density. Data are the mean ± s. e. m. (100 cells/condition in n = 3 independent experiments), two-tailed Mann-Whitney non-parametric test. Scale bar, 200 μm. b,c. Quantification of cytoplasmic rheology using genetically encoded multimeric nanoparticle (GEM)37. b)Time-and ensemble-averaged Mean Square Displacement (MSD) plots for control (blue line) and RAB5A (red line) in MCF10. DCIS. commonolayersseeded at jamming density. c)Cumulative distribution function showing the coefficient of diffusion, CDF, (D eff ) data of for control (blue line) and RAB5A (red line) MCF10.
Extended Data Fig. 6 | Fluidized MCF10. DCIS. com monolayers display an increase nuclear size and enrichment in PRC2-targets. a. Immunofluorescence images of nuclei in control (CTR) and RAB5A-(RAB5A)-MCF10. DCIS. commonolayers (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. comover 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. commonolayers. 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).
TAZ and cGAS-STING-dependency of EMP gene activation, collective migration and invasion in RAB5A fluidized monolayers. a. mRNA expression levels of CDH1, AXIN2, CDH2, EGF, MMP13, and ZEB1 in RAB5A-MCF10. DCIS. commonolayersovercontrolcells. Data are the mean ± s. d. (each dot represents an independent experiment). Values were normalized to the controls of each experiment. b. Phase-contrast images of control empty vector (CTR) and RAB5A-expressing (RAB5A) MCF10. DCIS. commonolayers(n= 5 independent experiments). Scale bar, 20 μm. c. Immunofluorescence images of doxycycline-treated control (CTR) and RAB5A-(RAB5A) MCF10. DCIS. comspheroids(arepresentativeimagefrom 5 independent experiments) stained as indicated. Scale bar 20 μm. d. Heatmap representing color-coded expression levels of differentially expressed EMT genes in RAB5A-MCF10.
DCIS. commonolayerssilencedforYAPandTAZ. The data are the ratio between the level of gene expression in each of the conditions tested relative to those of mock-treated RAB5A-cells. The mean ± s. d. (at least n = 3 independent experiments), two tailed Student's t-test (siRNA versus RAB5A-expressing cells) are reported in Source data_Figure 9D. e. mRNA expression levels of YAP and TAZ in RAB5A-MCF10. DCIS. comovercontrolcellssilencedwith the indicated oligos. Data are expressed as mean ± s. d. (n = 3 independent experiments). Values were normalized to the controls of each experiment. f. Heatmap representing color-coded expression levels of differentially expressed EMT genes in RAB5A-expressing MCF10. DCIS. commonolayerssilencedfor the indicated genes. This analysis was carried out on the same total mRNA from the experiment shown in Fig.
2a. The effectiveness of silencing is reported in Supplementary Fig. 4a. The mean ± s. d. (at least n = 3 independent experiments), two tailed Student's t-test (siRNA versus RAB5A-expressing cells) are reported in Source data_Figure 9F. g. Heatmap representing color-coded expression levels of differentially expressed EMT genes in RAB5A-MCF10. DCIS. commonolayerstreatedwith the following small molecules: cGAS inhibitor, 7 μg/ ml RU. 521, or the STING antagonist, 4 μg/ ml H-151. This analysis was carried out on the same total mRNA from the experiment shown in Fig. 2b. The data are the ratio between the level of gene expression in each of the conditions tested relative to vehicle-treated RAB5A-expressing cells. The mean ± s. d. (at least n = 3 independent experiments) with two tailed Student's t-test are reported in Source data_Figure 9G.
h. Scatter plot of the speed of wound closure of control (CTR) and RAB5A-expressing MCF10. DCIS. commonolayerstreatedwithvehicleor the cGAS inhibitor, 7 μg/ ml RU. 521, or the STING antagonist, 4 μg/ ml H-151 (SupplementaryVideo 11). Data are the mean ± s. d. (n = 3 independent experiments, in which 5 FOV/condition/experiment were analysed), two-tailed Mann-Whitney non-parametric test. i. Scatter plot of control (CTR) and RAB5A-expressing MCF10. DCIS. commonolayersinvadinginto a Matrigel coated wound and treated with vehicle or the cGAS inhibitor, 7 μg/ ml RU. 521, or the STING antagonist, 4 μg/ ml H-151 (SupplementaryVideo 12). Data are the mean ± s. d. (n = 3 independent experiments, in which 5 FOV/condition/ experiment were analysed), two-tailed Mann-Whitney non-parametric test, is indicated in the figure.
j. Heatmap representing color-coded expression levels of differentially expressed CytoDR genes in RAB5A-MCF10. DCIS. commonolayersshown in Supplementary Fig. 10ftreated with the indicated small molecules: cGAS inhibitor 7 μg/ ml RU. 521 or the STING antagonist 4 μg/ ml H-151. The data are the ratio between the level of gene expression in each of the conditions tested relative to vehicle-treated RAB5A-expressing cells. The mean ± s. d. (n> 3 independent experiments) and two tailed Student's t-test are reported in Source data_Figure 9J. P values are indicated in each graph.
, 1999; Lobet et al. , 2015)  (Figure  1B ). Microscopic observations indicated that diameters of the primary and lateral roots were fairly homogeneous within each branching order but were reduced by 30-40% between successive orders, varying from 188 ± 22 µm (± SD; n=64) in the primary root to as low as 69 µm in second order lateral roots. Thus, external root surfaces were calculated in the MTGs, assuming that root diameter was constant for a given root order, with a 30% decrease between successive root orders. Based on these detailed RSA representations, we developed a hydraulic model to simulate water fluxes in the corresponding segment network. In this model, called HydroRoot (https://github. com/openalea/hydroroot), each elementary root segment is associated with a derivative microcircuit containing both radial (k) and axial (K) hydraulic conductances.