Raw Data and Analysis for "Packing-Driven Mechanotransduction: local crowding overrides adhesion and stiffness cues for YAP Activation in Cellular Collectives"

Data Analysis and Segmentation Workflow Introduction This repository contains raw imaging data along with segmentation and analysis workflows. The data includes multiple repeats from different experimental conditions and is organized into folders for easy access. The provided code allows for segmentation, analysis, and visualization of the data. Folder Structure •SubstrateStiffness: Contains three conditions: Glass, 15 kPa and 1.5 kPa (WT MDCK cells). – .czi files raw data, 2D EPI-fluorescence images from three experimental repeats. –The images are simple 2D and contain the following channels: ∗F-actin (phalloidin) ∗Nucleus (Hoechst) ∗YAP (Immunostaining) – .npy files: Nuclear segmentation data for each image. – Excel File: Contains the following tables: ∗Table YAP nucl: Nuclear YAP ∗Table YAP doughnut: Cytoplasmic YAP ∗Table statistics LD: Local packing fraction ∗Table YAP voronoi: Cell area – Analysis: The analysis and segmentation can be performed using the Segmentation and Analysis of 2Ddata.ipynb notebook. •Blebbistatin: Contains two conditions: control cells (WT MDCK cells) and the cells that have been treated with Blebbistatin. – .nd2 files: raw data, 2D EPI-fluorescence images from three experimental repeats. –Contain the following channels: ∗YAP (Immunostaining) 1 ∗F-actin (Phalloidin) ∗Nucleus (Hoechst) – .npy files: Nuclear segmentation data for each image. – Excel File: Contains the following tables: ∗Table YAP nucl: Nuclear YAP ∗Table YAP doughnut: Cytoplasmic YAP ∗Table statistics LD: Local Packing Fraction ∗Table YAP voronoi: Cell area – Analysis: The analysis and segmentation can be performed using the Segmentation and Analysis of 2Ddata.ipynb notebook. •WT EcadKO LatA Y27632: –Contains 4 folders: WT,EcadKO,LatA, and Y27632. –Each folder contains 3 repeats of .nd2 Z-stack images (3D). –Channels include: ∗YAP (Immunostaining) ∗E-cadherin (Immunostaining) ∗F-actin (Phalloidin) ∗Nuclei (Hoechst) – Segmentation: The segmented files are large and thus are not provided. They can be created by using the 1 Segmentation 3D.ipynb notebook. – Analysis: The segmented data can be analyzed using the 2 Analysis 3D.ipynb notebook. – Excel Files: Each folder contains an Excel file with the required dataframes to reproduce the plots: ∗The dataframes can be used in 3 PrepareForPlotting 3D.ipynb to extract necessary properties such as YAP nuclear/cytoplasmic ratio, local packing fraction, and nuclear shape properties. There the properties are converted to microns for plotting. •SegmentationExample: –Contains one example of a 3D segmentation that can be applied in the 2 Analysis 3D.ipynb notebook for testing. •MonolayerStressMicroscopy: Contains two phase-contrast time-lapses of MDCK cells (two parts of one long), xml files with segmented and tracked cells, spots files with cell-centroid data and mat files that contain local density data, isotropic stress data etc. 2