Work Description

July 31, 2024 TITLE: Raw data (CSVs and pipelines) for Cell Painting and beta catenin immunofluorescence in MCF10A cells exposed to common chemical exposures AUTHORS: A. Tapaswi, N. Cemalovic, K. Polemi, J. Sexton, and J. Colacino CONTACT: Justin A Colacino, colacino@umich.edu SUPPORT: This work was supported by grants from the National Institutes of Health (R01 ES028802, T32 ES00706, P30 ES017885, R01 AG072396, P30 CA046592, S10 OD034245) and by the University of Michigan Rogel Cancer Center. KEY POINTS: a. In this study we are looking at the morphological perturbations when non-tumorigenic MCF10A cells were dosed with small molecules and a curated set of NHANES environmental chemicals. b. We observed significant differences in cellular phenotypes when subjected to a dose curve of the chosen toxicants. c. Following up on a small molecule-NHANES chemical pair, we compared the Wnt promotor CHIR to a organopesticide p,p�-DDE to understand the mode of action in potential breast cancer initiation. DESCRIPTION: MCF10A non-tumorigenic breast cells were dosed with environmental toxicants and stained with multiple cellular stains to study morphological perturbations. Following up on feature results, MCF10A cells were stained with an anti-beta catenin antibody to study beta catenin nuclear translocation. Cell profiler software was used to measure and export per cell data .CSV formats to be further analyze din BMDExpress2 and R studio SOFTWARE: a. Raw .CSV files can be opened in R to analyze. b. Cell profiler pipelines can be opened in open source Cell Profiler version 4.2.1 c. For QC analysis, .properties files can be opened in the open source Cell Profiler analyst version d. Post feature extraction data can be analyzed in BMDexpress2 METHOD: Post dosing and staining the cells with cellular stains we used the CellInsight CX5 microscope for automated image capture. Thermo Scientific HCS Studio: Cellomics Scan Version 6.6.1 was used to collect the images in .C01 format. The raw images were run through an illumination correction pipeline in Cell Profiler, which corrects for any variation in image background pixel intensities. Next, the raw images were run through a QC pipeline to collect blurriness and saturation cut off values. Finally, the raw images along with Illumination images were run in the Analysis pipeline. The Analysis pipeline was set to apply the illumination corrections, flag images based on the blurriness and saturation cut off values. Next, the primary, secondary and tertiary objects were delineated. In the following steps, the morphological feature was collected which included but were not limited to object size, shape, intensity, gradient, texture and correlation. All the raw data was exported in a .CSV format for further evaluation. FILES CONTAINED HERE: The folder structure for the experiments conducted are set up with the following file types. a. Raw .CSV files containing object and image level data for the controls and toxicant doses. b. Cell profiler pipelines run for every experiment (.cpproj file type). c. R studio code used to analyze the raw .CSV files. 1. 5Aza_ Chir_Cyclo_FCCP_PMA_Y a. The folder contains the following raw files: i. Cells: The .csv files captures all the measured features within the whole cell ii. Cytoplasm: The .csv files captures all the measured features within the cytoplasmic region of the cell iii. Image: The .csv file provides a well level data for all the measured features iv. Experiment: The .csv file give an overview of the software setup v. Nuclei: The .csv files captures all the measured features within the nuclear region of the cell vi. Metadata: This .csv file provides the information regarding the chemicals tested, doses used and replicates. b. The subfolder BMDexpress contains the per chemical text files generated to run through BMDexpress for downstream analysis c. The folder also contains the cellprofiler pipelines used to run the data analysis 2. BPA_BPS_BPF a. The folder contains the following raw files: i. Cells: The .csv files captures all the measured features within the whole cell ii. Cytoplasm: The .csv files captures all the measured features within the cytoplasmic region of the cell iii. Image: The .csv file provides a well level data for all the measured features iv. Experiment: The .csv file give an overview of the software setup v. Nuclei: The .csv files captures all the measured features within the nuclear region of the cell vi. Metadata: This .csv file provides the information regarding the chemicals tested, doses used and replicates. b. The subfolder BMDexpress contains the per chemical text files generated to run through BMDexpress for downstream analysis c. The folder also contains the cellprofiler pipelines used to run the data analysis 3. CFPO_DDT_DDE_Metalaxyl a. The folder contains the following raw files: i. Cells: The .csv files captures all the measured features within the whole cell ii. Cytoplasm: The .csv files captures all the measured features within the cytoplasmic region of the cell iii. Image: The .csv file provides a well level data for all the measured features iv. Experiment: The .csv file give an overview of the software setup v. Nuclei: The .csv files captures all the measured features within the nuclear region of the cell vi. Metadata: This .csv file provides the information regarding the chemicals tested, doses used and replicates. b. The subfolder BMDexpress contains the per chemical text files generated to run through BMDexpress for downstream analysis c. The folder also contains the cellprofiler pipelines used to run the data analysis 4.CTPB_Forskolin_Decitabine_DES_PGE2_3DZP a. The folder contains the following raw files: i. Cells: The .csv files captures all the measured features within the whole cell ii. Cytoplasm: The .csv files captures all the measured features within the cytoplasmic region of the cell iii. Image: The .csv file provides a well level data for all the measured features iv. Experiment: The .csv file give an overview of the software setup v. Nuclei: The .csv files captures all the measured features within the nuclear region of the cell vi. Metadata: This .csv file provides the information regarding the chemicals tested, doses used and replicates. b. The subfolder BMDexpress contains the per chemical text files generated to run through BMDexpress for downstream analysis c. The folder also contains the cellprofiler pipelines used to run the data analysis 5.E2_Methomyl_Imicloprid_Thiacloprid a. The folder contains the following raw files: i. Cells: The .csv files captures all the measured features within the whole cell ii. Cytoplasm: The .csv files captures all the measured features within the cytoplasmic region of the cell iii. Image: The .csv file provides a well level data for all the measured features iv. Experiment: The .csv file give an overview of the software setup v. Nuclei: The .csv files captures all the measured features within the nuclear region of the cell vi. Metadata: This .csv file provides the information regarding the chemicals tested, doses used and replicates. b. The subfolder BMDexpress contains the per chemical text files generated to run through BMDexpress for downstream analysis c. The folder also contains the cellprofiler pipelines used to run the data analysis 6.FG4592_Thapsigargin_Menadione a. The folder contains the following raw files: i. Cells: The .csv files captures all the measured features within the whole cell ii. Cytoplasm: The .csv files captures all the measured features within the cytoplasmic region of the cell iii. Image: The .csv file provides a well level data for all the measured features iv. Experiment: The .csv file give an overview of the software setup v. Nuclei: The .csv files captures all the measured features within the nuclear region of the cell vi. Metadata: This .csv file provides the information regarding the chemicals tested, doses used and replicates. b. The subfolder BMDexpress contains the per chemical text files generated to run through BMDexpress for downstream analysis c. The folder also contains the cellprofiler pipelines used to run the data analysis 7.Lead_Arsenic_Mercury_Copper_Cadmium a. The folder contains the following raw files: i. Cells: The .csv files captures all the measured features within the whole cell ii. Cytoplasm: The .csv files captures all the measured features within the cytoplasmic region of the cell iii. Image: The .csv file provides a well level data for all the measured features iv. Experiment: The .csv file give an overview of the software setup v. Nuclei: The .csv files captures all the measured features within the nuclear region of the cell vi. Metadata: This .csv file provides the information regarding the chemicals tested, doses used and replicates. b. The subfolder BMDexpress contains the per chemical text files generated to run through BMDexpress for downstream analysis c. The folder also contains the cellprofiler pipelines used to run the data analysis 8.PFDA_PFNA a. The folder contains the following raw files: i. Cells: The .csv files captures all the measured features within the whole cell ii. Cytoplasm: The .csv files captures all the measured features within the cytoplasmic region of the cell iii. Image: The .csv file provides a well level data for all the measured features iv. Experiment: The .csv file give an overview of the software setup v. Nuclei: The .csv files captures all the measured features within the nuclear region of the cell vi. Metadata: This .csv file provides the information regarding the chemicals tested, doses used and replicates. b. The subfolder BMDexpress contains the per chemical text files generated to run through BMDexpress for downstream analysis c. The folder also contains the cellprofiler pipelines used to run the data analysis 9.Thiram_25DCP_14DCB_BPA_MPB_PPB a. The folder contains the following raw files: i. Cells: The .csv files captures all the measured features within the whole cell ii. Cytoplasm: The .csv files captures all the measured features within the cytoplasmic region of the cell iii. Image: The .csv file provides a well level data for all the measured features iv. Experiment: The .csv file give an overview of the software setup v. Nuclei: The .csv files captures all the measured features within the nuclear region of the cell vi. Metadata: This .csv file provides the information regarding the chemicals tested, doses used and replicates. b. The subfolder BMDexpress contains the per chemical text files generated to run through BMDexpress for downstream analysis c. The folder also contains the cellprofiler pipelines used to run the data analysis 10.TSA_VPA_Etoposide_JQ1_Tunicamycin a. The folder contains the following raw files: i. Cells: The .csv files captures all the measured features within the whole cell ii. Cytoplasm: The .csv files captures all the measured features within the cytoplasmic region of the cell iii. Image: The .csv file provides a well level data for all the measured features iv. Experiment: The .csv file give an overview of the software setup v. Nuclei: The .csv files captures all the measured features within the nuclear region of the cell vi. Metadata: This .csv file provides the information regarding the chemicals tested, doses used and replicates. b. The subfolder BMDexpress contains the per chemical text files generated to run through BMDexpress for downstream analysis c. The folder also contains the cellprofiler pipelines used to run the data analysis 11. Beta_Catenin_immunofluorescence experiment a. The folder contains the following raw files: i. MyExpt_Cells: The .csv files captures all the measured features within the whole cell ii. MyExpt_Cytoplasm: The .csv files captures all the measured features within the cytoplasmic region of the cell iii. MyExpt_Image: The .csv file provides a well level data for all the measured features iv. MyExpt_FilteredNuclei: The .csv files captures all the measured features within the nuclear region of the cell v. Metadata: This .csv file provides the information regarding the chemicals tested, doses used and replicates. b. The folder also contains the cellprofiler pipelines used to run the data analysis c. The folder also contains the R code run to get the data visualizations 12. Cell_counts_analysis a.The subfolder Nhanes contains: i. The raw cell counts files for all the chemicals tested ii. The R code to get the p values and generate heatmaps iii. The .csv files for cell counts transposed data and the .csv files containing p value in * format needed for heatmap generation b.The subfolder Small molecules contains: i. The raw cell counts files for all the chemicals tested ii. The R code to get the p values and generate heatmaps iii. The .csv files for cell counts transposed data and the .csv files containing p value in * format needed for heatmap generation 13. Illumination pipeline a. This is a cell profiler pipeline which can be used to perform illumination correction in images. b. The pipeline is common across experiments, and only images that need to the analysed need to be swapped in the images module of the pipeline. 14. QC and Processing Mega Integration - 072624 a. This is the R code use to analyze the cell painting raw data and generation of BMDexpress input files. 15. Cell Painting Tox In Vitro 7-31-24.bm2 a. This .bm2 file is a project file that can be opened in BMDExpress to look at the data for each of the chemicals.