Data from: Nuclear blebs are associated with destabilized chromatin-packing domains
Data files
Aug 05, 2026 version files 12.94 MB
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ctrl_phi.csv
11.77 KB
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DMSO_bleb.npy
320 B
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DMSO_BODY.npy
26.80 KB
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Lamin_bleb.npy
280 B
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Lamin_Body.npy
1.42 KB
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LMNB1B2_Treatments_CTCF_8-11-23_CSV.zip
451.77 KB
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LMNB1B2_Treatments_CTCF_8-11-23.xlsx
560.75 KB
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LMNB1B2_Treatments_CTCF_9-6-23_CSVs.zip
264.73 KB
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LMNB1B2_Treatments_CTCF_9-6-23.xlsx
381.59 KB
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Nuclear_Bleb_Check_Treatments_LMNB1B2_2-1-24.xlsx
27.41 KB
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Nuclear_Bleb_Check_Treatments_LMNB1B2_8-11-23.xlsx
29.38 KB
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Nuclear_Bleb_Check_Treatments_LMNB1B2_9-6-23.xlsx
28.58 KB
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README.md
15.70 KB
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Replotting_Bleb_PWS_and_PWS_Dynamics_Data_CSVs.zip
347.50 KB
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Replotting_Bleb_PWS_and_PWS_Dynamics_Data.xlsx
292.29 KB
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Result_of_Control_Result_of_dyn-1-zoom_BLUE.avi
527.16 KB
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Result_of_TSA_Result_of_dyn-1-zoom_BLUE.avi
441.94 KB
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summary_charts_for_LMNB1B2_Treatments_CTCF_8-11-23.pdf
52.42 KB
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TSA_BLEB.npy
272 B
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TSA_BODY.npy
17.99 KB
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tsa_phi.csv
6.06 KB
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Unformatted_XLSX_Files.zip
25.46 KB
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Vid_S3_(1).avi
9.43 MB
Abstract
Disrupted nuclear shape is associated with multiple pathological processes, including premature aging disorders, cancer-relevant chromosomal rearrangements, and DNA damage. Nuclear blebs (i.e., herniations of the nuclear envelope) have been induced by (1) nuclear compression, (2) nuclear migration (e.g., cancer metastasis), (3) actin contraction, (4) lamin mutation or depletion, and (5) heterochromatin enzyme inhibition. Recent work has shown that chromatin transformation is a hallmark of bleb formation, but the transformation of higher-order structures in blebs is not well understood. As higher-order chromatin has been shown to assemble into nanoscopic packing domains, we investigated whether (1) packing domain organization is altered within nuclear blebs and (2) alteration in packing domain structure contributed to bleb formation. Using Dual-Partial Wave Spectroscopic microscopy, we show that chromatin packing domains within blebs are transformed both by B-type lamin depletion and the inhibition of heterochromatin enzymes compared to the nuclear body. Pairing these results with single-molecule localization microscopy of constitutive heterochromatin, we show fragmentation of nanoscopic heterochromatin domains within bleb domains. Overall, these findings indicate that chromatin within blebs is associated with a fragmented higher-order chromatin structure.
https://doi.org/10.5061/dryad.gb5mkkx0d
Description of the data and file structure
Imaging Data
All imaging data can be sent upon request. This repository holds the PWS Imaging and PWS Dynamics FMM and Diffusion raw values in the Excel file. STORM Data can also be sent upon request, but a raw numpy file has been provided for ease of use. This holds all of the identified heterochromatic clusters and their size. CTCF measurements from confocal data are stored in csv files in this repository.
Files and variables
File: Result_of_Control_Result_of_dyn-1-zoom_BLUE.avi
Description: Result Image for PWS Dynamics for control cell
File: Result_of_TSA_Result_of_dyn-1-zoom_BLUE.avi
Description: Result Image for PWS Dynamics for TSA-treated cell
File: DMSO_bleb.npy
Description: DMSO Bleb SMLM cluster size data in .npy. Each entry in the array represents the size of one cluster normalized to the area of a circle with a radius of 80 nm.
File: Vid_S3_(1).avi
Description: PWS Dynamics Example Video
File: Lamin_bleb.npy
Description: Lamin Bleb SMLM cluster size data in .npy. Each entry in the array represents the size of one cluster normalized to the area of a circle with a radius of 80 nm.
File: Lamin_Body.npy
Description: Lamin Body SMLM cluster size data in .npy. Each entry in the array represents the size of one cluster normalized to the area of a circle with a radius of 80 nm
File: ctrl_phi.csv
Description: CVC data for Control Condition
Variables
- phi_bleb: Data for bleb
- phi_body: Data for nuclear body
- phi_bbratio: Data for bleb/nuclear body ratio
File: tsa_phi.csv
Description: CVC data for TSA treatment case
Variables
- phi_bleb: Data for bleb
- phi_body: Data for nuclear body
- phi_bbratio: Data for bleb/nuclear body ratio
File: DMSO_BODY.npy
Description: DMSO Body SMLM cluster size data in .npy. Each entry in the array represents the size of one cluster normalized to the area of a circle with a radius of 80 nm.
File: TSA_BODY.npy
Description: TSA Body SMLM cluster size data in .npy. Each entry in the array represents the size of one cluster normalized to the area of a circle with a radius of 80 nm.
File: TSA_BLEB.npy
Description: TSA BlebSMLM cluster size data in .npy. Each entry in the array represents the size of one cluster normalized to the area of a circle with a radius of 80 nm.
File: Nuclear_Bleb_Check_Treatments_LMNB1B2_8-11-23.xlsx and Unformatted_XLSX_Files.zip
Description: Bleb frequency across treatme,nts replicate 1. Within this file, we have separated the raw counts in one sheet with the percentages for that given replicate in another sheet. The zip folder has the unformatted Excel files with the same contents.
Sheet Key:
- Raw_Count: Raw bleb count sheet
- Percentages: Percentage of blebs. Data from sheet 1.
The colored label above the categories (#bleb, #micronuclei...) represents the drug treatment for that condition. It is repeated for each column for ease of interpretation with codto justver, we encourage you just to use the already generated averages and percentages table in the second sheet of this Excel file. The raw counts are kept for completeness. Colors for treatments of green and red categories were used for ease of viewing to create contrast and hold no meaning.
Column Key Raw Counts:
- Cell #: FOV acquisition identifier, not indicative of actual individual cells but rather the FOV number where the nuclei were counted
- # Nuclei: Number of nuclei in that Cell# FOV
- # Blebs: Number of blebs in that Cell# FOV
- #MN: number of micronuclei in that Cell# FOV
- # Ruptures: number of ruptures in that Cell# FOV
Note: this is repeated for the different treatments. Each treatment is colored as a different color since the columns are sequential. As such, the data column key above repeats.
Column Key Percentages:
- DMSO1-3 indicate the three replicates of the DMSO CTRL group
- 24 hr GSK343: 24 Hour GSK343 treatment
- 24 hr Auxin + GSK343: dual treatment of auxin and GSK343
- 24 hr TSA: 24-hour TSA
- 24hr Auxin + TSA: dual treatment of auxin and TSA
Note: Row names in the first column are the same as the column names in the raw count sheet.
- % Deformed nuclei is any nuclei that has a bleb, micronuclei, or is ruptured.
File: Nuclear_Bleb_Check_Treatments_LMNB1B2_9-6-23.xlsx and Unformatted_XLSX_Files.zip
Description: Bleb frequency across treatments replicate 1. Within this file,e we have separated the raw counts in one sheet with the percentages for that given replicate in another sheet. The zip folder has the unformatted Excel files with the same contents.
Sheet Key:
- Raw_Count: Raw bleb count sheet
- Percentages: Percentage of blebs. Data from sheet 1.
The colored label above the categories (#bleb, #micronuclei...) represents the drug treatment for that condition. It is repeated for each column for ease of interpretation with codto justver, we encourage you just to use the already generated averages and percentages table in the second sheet of this Excel file. The raw counts are kept for completeness. Colors for treatments of green and red categories were used for ease of viewing to create contrast and hold no meaning.
Column Key Raw Counts:
- Cell #: FOV acquisition identifier, not indicative of actual individual cells but rather the FOV number where the nuclei were counted
- # Nuclei: Number of nuclei in that Cell# FOV
- # Blebs: Number of blebs in that Cell# FOV
- #MN: number of micronuclei in that Cell# FOV
- # Ruptures: number of ruptures in that Cell# FOV
Note: this is repeated for the different treatments. Each treatment is colored as a different color since the columns are sequential. As such, the data column key above repeats.
Column Key Percentages:
- DMSO1-3 indicate the three replicates of the DMSO CTRL group
- 24 hr GSK343: 24 Hour GSK343 treatment
- 24 hr Auxin + GSK343: dual treatment of auxin and GSK343
- 24 hr TSA: 24-hour TSA
- 24hr Auxin + TSA: dual treatment of auxin and TSA
Note: Row names in the first column are the same as the column names in the raw count sheet.
- % Deformed nuclei is any nuclei that has a bleb, micronuclei, or is ruptured.
File: Nuclear_Bleb_Check_Treatments_LMNB1B2_2-1-24.xlsx and Unformatted_XLSX_Files.zip
Description: Bleb frequency across treatme,nts replicate 3. Within this file, we have separated the raw counts in one sheet with the percentages for that given replicate in another sheet. The zip folder has the unformatted Excel files with the same contents.
Sheet Key:
- Raw_Count: Raw bleb count sheet
- Percentages: Percentage of blebs. Data from sheet 1.
The colored label above the categories (#bleb, #micronuclei...) represents the drug treatment for that condition. It is repeated for each column for ease of interpretation; however, we encourage you just to use the already generated averages and percentages table in the second sheet of this Excel file. The raw counts are kept for completeness. Colors for treatments of green and red categories were used for ease of viewing to create contrast and hold no meaning.
Column Key Raw Counts:
- Cell #: FOV acquisition identifier, not indicative of actual individual cells but rather the FOV number where the nuclei were counted
- # Nuclei: Number of nuclei in that Cell# FOV
- # Blebs: Number of blebs in that Cell# FOV
- #MN: number of micronuclei in that Cell# FOV
- # Ruptures: number of ruptures in that Cell# FOV
Note: this is repeated for the different treatments. Each treatment is colored as a different color since the columns are sequential. As such, the data column key above repeats.
Column Key Percentages:
- DMSO1-3 indicate the three replicates of the DMSO CTRL group
- 24 hr GSK343: 24 Hour GSK343 treatment
- 24 hr Auxin + GSK343: dual treatment of auxin and GSK343
- 24 hr TSA: 24-hour TSA
- 24hr Auxin + TSA: dual treatment of auxin and TSA
Note: Row names in the first column are the same as the column names in the raw count sheet.
- % Deformed nuclei is any nuclei that has a bleb, micronuclei, or is ruptured.
File: LMNB1B2_Treatments_CTCF_8-11-23.xlsx and LMNB1B2_Treatments_CTCF_8-11-23_CSV.zip
Description: CTCF measurements across different treatments presented in the manuscript. We report the CTCF values for H3K27me3 and H3K27ac for the different treatments. Average CTCF for each condition across all cells was reported as the final column. The cell column represents the image FOV we used for each acquisition. The ROI # here represents the number of nuclei being analyzed within this FOV. We apologize for the unusual nomenclature, but this was built into the analysis software we use, so cell just means image, and ROI# refers to the number of nuclei in that FOV. The sheets in this Excel file are split by treatment and label and indicated by name. For example, the first sheet given is Control_H3K27me3, indicating that this is the data for the control group labeled for H3K27me3. As a reminder to the reader, the treatments are
- No Treatment (Control)
- Auxin Degradation of Lamin B1/B2 (Auxin)
- GSK treatments (GSK343 treatment for EZH2i)
- TSA treatment (Trichostatin A treatment for HDACi)
- Auxin + GSK (dual treatment of Auxin and GSK343)
- Auxin + TSA (dual treatment of Auxin and TSA)
Note: There are two sheets per aforementioned point, one for H3K27me3 and one for H3K27ac.
The following Column key is for the sheets that house the data, not summary sheets. The zip folder has the unformatted CSV files with the same contents.
Column Key:
- Cell: FOV identifier, not an actual cell but the FOV count during acquisition. Cell 1 means the first image taken
- ROI#: Number identifier for a given nucleus within a FOV
- ROI Name: Name of the ROI as saved by acquisition Software
- Area: Area of nucleus in um^2
- Mean: the average intensity reading for that given nucleus
- Min: the minimum intensity reading
- Max: the maximum reading
- X/Y: coordinates of the ROI
- IntDen: Integrated Density, the product of the area and mean value
- RawIntDen: Raw Integrated Density, the sum of pixel intensity values in the nuclear ROI
- Average CTCF: the average CTCF across all nuclei in the sheet.
Note: Highlighted Background cells indicate that these are background measurements within that FOV
Column Key for Summary Sheet:
- Control H3K27me3: H3k72me3 CTCF Control readings
- Auxin H3K27me3: H3k72me3 CTCF Auxin treated group readings
- Control H3K27me3: H3k72ac CTCF Control readings
- Auxin H3K27me3: H3k72ac CTCF Auxin treated group readings
Note: Sheet 1 is a test sheet with CTCF measurements across different channels. Column titles are the same as the treatment data sheets.
File: summary_charts_for_LMNB1B2_Treatments_CTCF_8-11-23.pdf
Description: PDF of summary results from the LMNB1B2 8-11-23 replicates. There are two plots in total for each label: H3K27me3 and H3K27ac. The summary plots include the data for the control and Auxin degradation case.
File: LMNB1B2_Treatments_CTCF_9-6-23.xlsx and LMNB1B2_Treatments_CTCF_9-6-23_CSVs.zip
Description: CTCF measurements across different treatments presented in the manuscript. We report the CTCF values for H3K27me3 and H3K27ac for the different treatments. Average CTCF for each condition across all cells was reported as the final column. The cell column represents the image FOV we used for each acquisition. The ROI # here represents the number of nuclei being analyzed within this FOV. We apologize for the unusual nomenclature, but this was built into the analysis software we use, so cell just means image, and ROI# refers to the number of nuclei in that FOV. The sheets in this Excel file are split by treatment and label and indicated by name. For example, the first sheet given is Control_H3K27me3, indicating that this is the data for the control group labeled for H3K27me3. As a reminder to the reader, the treatments are
- No Treatment (Control)
- Auxin Degradation of Lamin B1/B2 (Auxin)
- GSK treatments (GSK343 treatment for EZH2i)
- TSA treatment (Trichostatin A treatment for HDACi)
- Auxin + GSK (dual treatment of Auxin and GSK343)
- Auxin + TSA (dual treatment of Auxin and TSA)
Note: There are two sheets per aforementioned point, one for H3K27me3 and one for H3K27ac.
The following Column key is for the sheets that house the data, not summary sheets. The zip folder has the unformatted CSV files with the same contents.
Column Key:
- Cell: FOV identifier, not an actual cell but the FOV count during acquisition. Cell 1 means the first image taken.
- ROI#: Number identifier for a given nucleus within a FOV
- ROI Name: Name of the ROI as saved by acquisition Software
- Area: Area of nucleus in um^2
- Mean: the average intensity reading for that given nucleus
- Min: the minimum intensity reading
- Max: the maximum reading
- X/Y: coordinates of the ROI
- IntDen: Integrated Density, the product of the area and mean value
- RawIntDen: Raw Integrated Density, the sum of pixel intensity values in the nuclear ROI
- Average CTCF: the average CTCF across all nuclei in the sheet.
Note: Highlighted Background cells indicate that these are background measurements within that FOV.
File: Replotting_Bleb_PWS_and_PWS_Dynamics_Data.xlsx and Replotting_Bleb_PWS_and_PWS_Dynamics_Data_CSVs.zip
Description: PWS Dynamics and PWS data
The zip folder has the unformatted CSV files with the same contents. PWS refers to Partial Wave Spectroscopy method. Each column details the experimental grouping and the replicate. The data is split into two regions, either the body or the bleb. Where the first row indicates the location of this data by appending it to the end as follows:
- Control: data for control group body
- Control_blebs: data for control blebs
The treatment groups are also indicated by the columns in the first row. The list is as follows:
- Control: No Treatment
- 24 Hour Auxin: Auxin degradation of Lamin
Beneath the column indicator of location and treatment is an indicator for the replicate ID. This is used to report the mean and standard deviation for each group in the yellow highlighted columns.
There are different sheets that correspond to the different data we get from PWS. The keys for each sheet are below. The left-hand of the data corresponds to the raw replicates, and the highlighted portion refers to the average for all replicates for the conditions.
Keys:
- D: Chromatin Packing Scaling D
- Diff: Diffusion Coefficient
- FMM: Fractional Moving Mass
For more information: https://www.nature.com/articles/s41467-019-09717-6
Note: The column names are repeated to indicate the treatment type and keep column names short to avoid including long replication indicators in the same columns.
Code/software
In-house developed Python code used for SMLM analysis. For any questions regarding use, please reach out to nicolasacosta2026@u.northwestern.edu.
Data and Image Analysis
We used GraphPad Prism 10.1.1 for making all plots. For immunofluorescence imaging, maximum-intensity projections of Z-series images were generated using FIJI. To quantify nuclear bleb frequency, we defined blebs as herniations still connected to the nuclear body. We considered ruptures to be cells that were no longer intact, and we considered micronuclei to be herniations that were no longer connected to the nuclear body and of similar sizes to nuclear blebs. For each field of view, the number of nuclei and the number of each nuclear bleb type were manually counted using FIJI. We then determined the percentages of total cells within each tested condition that displayed each nuclear bleb type.
Super-resolution Data Analysis:
We used the Thunder-STORM FIJI Plug-in to apply Maximum Likelihood Estimation fitting of a gaussian point spread function to our image stack. Localization datasets were then put into our Python script that utilized DBSCAN (epsilon=50, min_pts=3) to cluster our localized heterochromatic events. Heterochromatin domain size was estimated by fitting a polygon to the peripheral cluster points using the scipy Convex Hull** method. Outlier clusters smaller than twice the mean uncertainty of our localization (~ 25 nm) or larger than 800 nm were removed from the analysis. Results displayed are concatenations of identified heterochromatic domains across all cells in that condition.
Statistical Analysis and Quantification
Statistical analysis was performed using GraphPad Prism 10.1.1 and Microsoft Excel. Pairwise comparisons were calculated on datasets consisting of, at a minimum, biologically independent duplicate samples using a two-tailed unpaired t test or Mann-Whitney test. The type of statistical test is specified in each case. Experimental data are presented either the mean ± SEM or mean ± SD, as stated in figure legends. A P value of < 0.05 was considered significant. Statistical significance levels are denoted as follows: n.s. = not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Sample numbers (# of nuclei, n), the number of replicates (N), and the type of statistical test used is indicated in figure legends.