DOI: 10.5061/dryad.mcvdnckff
title: Data from: Reversibility of nuclear and 3D Genomic changes in non-cancerous fibroblasts after constricted migration

description: Metastatic cancer cells and healthy fibroblasts must traverse through
 tight constrictive spaces to reach secondary sites. We have previously
 shown that human melanoma (A375) cells that pass through multiple
 constricted migrations experience stable changes to their nucleus
 morphology and 3D genome structures and undergo a stable phenotype
 transition from a low to high migratory state. Here, we investigate
 whether fibroblasts (BJ-5ta), which are non-cancerous and have an inherent
 ability to migrate to fulfill roles in wound repair, experience nuclear
 and 3D genomic changes with constricted migration. While BJ-5ta cells do
 not get progressively better at migrating with sequential attempts,
 confocal microscopy analysis of cells stained for Lamin A/C and DAPI after
 migration shows that these cells do experience nuclear deformations after
 constricted migration. Unlike the stable changes observed in cancer cells,
 however, these deformations recovered back to unmigrated levels following
 proliferation and cell movement and did not induce fibrotic smooth muscle
 alpha actin expression in these cells. Our study shows that healthy
 migratory cells are not more robust against alterations caused by
 constricted migration but can recover from the ones that do arise more
 readily than cancer cells.
description: Constricted Migration Assays: Transwell
 filters with 5µm pores (VWR-10769-236) were used. The bottom of each
 filter was coated with 40µL of 10ug/µL fibronectin (Corning, 354008) for
 ~30 min. 100,000 BJ-5ta cells in 100µL of fully supplemented media were
 placed into the top well of the transwell with 500µL of fully supplemented
 media in the bottom chamber. Cells were allowed to attach overnight. The
 following day both the top and bottom chamber’s media were replaced with a
 4:1 ratio of DMEM and 1x Medium 199, but with 1% BSA instead of 10% FBS.
 Cells were starved overnight. The following day, the bottom chamber’s
 media was replaced with a 4:1 ratio of DMEM and Medium 199 supplemented
 with 20% FBS and 5 μg/mL of FGF (Gibco, PHG0368V).  
 For sequential rounds of migration, the migrated cells were
 seeded into a new well of a 24‐well plate to expand. When cells reached
 80–90% confluency, another Transwell migration was performed as previously
 described. Only the bottom cells were collected and continued to be used
 for sequential rounds of migration.  Immunofluorescent
 Confocal Imaging  Approximately 50,000 BJ-5ta cells for
 each condition were seeded into 35‐mm glass bottom dishes (MatTek,
 P35GC-1.5-10-C). Cells were allowed to attach overnight and then
 cross‐linked with 4% formaldehyde for 10 min followed by three, 5‐min
 washes with PBS. After washing, cells were permeabilized with
 permeabilization buffer (10% goat serum, 0.5% Triton in PBS) for 1 h at
 room temperature. After the incubation, primary antibody for LaminA/C
 (Santa Cruz, sc-376248), diluted in antibody dilution buffer (5% Goat
 serum, 0.25% Triton in PBS), was added and incubated overnight at 4°C.
 After primary antibody incubation, cells were washed three times with PBS
 for 5 min for each. Cells were then incubated with secondary antibodies
 (Alexa Fluor 488, Invitrogen, R37120) per manufacturer's directions
 (two drops of secondary antibody/1 mL of PBS) for 30 min at room
 temperature. After secondary antibody incubation, cells were washed three
 times with PBS and sealed using mounting media with DAPI (Invitrogen,
 P36962). Slides were treated in the mounting media for 24hr before
 imaging. Cells were imaged using a Leica Sp8 Confocal microscope equipped
 with a 63x oil immersion objective. For αSMA experiments, cells were
 seeded onto 24 well plate coverslips (Fisher Scientific, 50-194-4702) with
 either no treatment, starvation and FGF spike-in as described earlier,
 directly after 5um transwell migration, or after migration and allowed to
 recover for 48hrs. Cells were then fixed as above and stained with an αSMA
 primary antibody (Invitrogen, MA1-06110) overnight in 4C. Secondary
 antibody and washes were done as above. Cells were then imaged on a Leica
 Sp8 Confocal microscope with a 40x water immersion objective. For a
 positive control for αSMA induction, BJ-5ta cells were treated with 5ng/mL
 TGFbeta for 48hrs before fixing and staining as described above on 24 well
 plate sized coverslips. 
description: # Data from: Reversibility of nuclear and 3D Genomic changes in
 non-cancerous fibroblasts after constricted migration Dataset DOI:
 [10.5061/dryad.mcvdnckff](https://doi.org/10.5061/dryad.mcvdnckff) ##
 Description of the data and file structure ### Files and variables ####
 File: BJ5ta_Unmigrated_LaminAC_H3K9me3.lif **Description:** Control BJ-5ta
 cells stained for Lamin A/C (488, green) and H3K9me3 (594, red) and DAPI
 (blue, DNA). #### File: BJ5ta_StarveSpike_LaminAC_H3K9me3.lif
 **Description:** BJ-5ta cells in conditions mimicking preparation for
 migration: starvation in serum-free media overnight and then spiked with
 FGF for 24 hours, then stained for Lamin A/C (488, green) and H3K9me3
 (594, red) and DAPI (blue, DNA). #### File:
 BJ5ta_BR3_4days_LaminAC_H3K9me3.lif **Description:** BJ-5ta cells passed
 through 3 sequential rounds of 5 micron Transwell migration, then allowed
 to proliferate for 4 days in standard media. Stained for Lamin A/C (488,
 green) and H3K9me3 (594, red) and DAPI (blue, DNA). #### File:
 BJ5ta_BR3_3days_LaminAC_H3K9me3.lif **Description:** BJ-5ta cells passed
 through 3 sequential rounds of 5 micron Transwell migration, then allowed
 to proliferate for 3 days in standard media. Stained for Lamin A/C (488,
 green) and H3K9me3 (594, red) and DAPI (blue, DNA). #### File:
 BJ5ta_BR4_LaminAC_H3K9me3.lif **Description:** BJ-5ta cells passed through
 4 sequential rounds of 5 micron Transwell migration, then stained for
 Lamin A/C (488, green) and H3K9me3 (594, red) and DAPI (blue, DNA). ####
 File: BJ5ta_BR3_2days_LaminAC_H3K9me3.lif **Description:** BJ-5ta cells
 passed through 3 sequential rounds of 5 micron Transwell migration, then
 allowed to proliferate for 2 days in standard media. Stained for Lamin A/C
 (488, green) and H3K9me3 (594, red) and DAPI (blue, DNA). #### File:
 BJ5ta_BR3_1day_LaminAC_H3K9me3.lif **Description:** BJ-5ta cells passed
 through 3 sequential rounds of 5 micron Transwell migration, then allowed
 to proliferate for 1 day in standard media. Stained for Lamin A/C (488,
 green) and H3K9me3 (594, red) and DAPI (blue, DNA). #### File:
 BJ5ta_BR3_LaminAC_H3K9me3.lif **Description:** BJ-5ta cells passed through
 3 sequential rounds of 5 micron Transwell migration, then stained for
 Lamin A/C (488, green) and H3K9me3 (594, red) and DAPI (blue, DNA). ####
 File: BJ5ta_BR2_4days_LaminAC_H3K9me3.lif **Description:** BJ-5ta cells
 passed through 2 sequential rounds of 5 micron Transwell migration, then
 allowed to proliferate for 4 days in standard media. Stained for Lamin A/C
 (488, green) and H3K9me3 (594, red) and DAPI (blue, DNA). #### File:
 BJ5ta_BR2_3days_LaminAC_H3K9me3.lif **Description:** BJ-5ta cells passed
 through 2 sequential rounds of 5 micron Transwell migration, then allowed
 to proliferate for 3 days in standard media. Stained for Lamin A/C (488,
 green) and H3K9me3 (594, red) and DAPI (blue, DNA). #### File:
 BJ5ta_BR2_LaminAC_H3K9me3_01.lif **Description:** BJ-5ta cells passed
 through 2 sequential rounds of 5 micron Transwell migration, then stained
 for Lamin A/C (488, green) and H3K9me3 (594, red) and DAPI (blue, DNA).
 #### File: BJ5ta_BR2_2days_LaminAC_H3K9me3.lif **Description:** BJ-5ta
 cells passed through 2 sequential rounds of 5 micron Transwell migration,
 then allowed to proliferate for 2 days in standard media. Stained for
 Lamin A/C (488, green) and H3K9me3 (594, red) and DAPI (blue, DNA). ####
 File: BJ5ta_BR2_1day_LaminAC_H3K9me3.lif **Description:** BJ-5ta cells
 passed through 2 sequential rounds of 5 micron Transwell migration, then
 allowed to proliferate for 1 day in standard media. Stained for Lamin A/C
 (488, green) and H3K9me3 (594, red) and DAPI (blue, DNA). #### File:
 BJ5ta_BR2_LaminAC_H3K9me3.lif **Description:** BJ-5ta cells passed through
 2 sequential rounds of 5 micron Transwell migration, then stained for
 Lamin A/C (488, green) and H3K9me3 (594, red) and DAPI (blue, DNA). ####
 File: BJ5ta_BR1_4days_LaminAC_H3K9me3.lif **Description:** BJ-5ta cells
 passed through 1round of 5 micron Transwell migration, then allowed to
 proliferate for 4 days in standard media. Stained for Lamin A/C (488,
 green) and H3K9me3 (594, red) and DAPI (blue, DNA). #### File:
 BJ5ta_BR1_3days_LaminAC_H3K9me3.lif **Description:** BJ-5ta cells passed
 through 1round of 5 micron Transwell migration, then allowed to
 proliferate for 3 days in standard media. Stained for Lamin A/C (488,
 green) and H3K9me3 (594, red) and DAPI (blue, DNA). #### File:
 BJ5ta_BR1_2days_LaminAC_H3K9me3.lif **Description:** BJ-5ta cells passed
 through 1round of 5 micron Transwell migration, then allowed to
 proliferate for 2 days in standard media. Stained for Lamin A/C (488,
 green) and H3K9me3 (594, red) and DAPI (blue, DNA). #### File:
 BJ5ta_BR1_1day_LaminAC_H3K9me3.lif **Description:** BJ-5ta cells passed
 through 1round of 5 micron Transwell migration, then allowed to
 proliferate for 1 day in standard media. Stained for Lamin A/C (488,
 green) and H3K9me3 (594, red) and DAPI (blue, DNA). #### File:
 BJ5ta_BR1_LaminAC_H3K9me3_01.lif **Description:** BJ-5ta cells passed
 through 1round of 5 micron Transwell migration, then stained for Lamin A/C
 (488, green) and H3K9me3 (594, red) and DAPI (blue, DNA). #### File:
 BJ5ta_BR1_LaminAC_H3K9me3.lif **Description:** BJ-5ta cells passed through
 1round of 5 micron Transwell migration, then stained for Lamin A/C (488,
 green) and H3K9me3 (594, red) and DAPI (blue, DNA). #### File:
 BJ5ta_aSMA_Untreated.lif **Description:** Control BJ-5ta cells stained for
 smooth muscle alpha actin (488, green) and DAPI (blue, DNA). #### File:
 BJ5ta_aSMA_StarveSpike.lif **Description:** BJ-5ta cells in conditions
 mimicking preparation for migration: starvation in serum-free media
 overnight and then spiked with FGF for 24 hours, then stained for smooth
 muscle alpha actin (488, green) and DAPI (blue, DNA). #### File:
 BJ5ta_aSMA_BR1.lif **Description:** BJ-5ta cells passed through 1round of
 5 micron Transwell migration, then stained for smooth muscle alpha actin
 (488, green) and DAPI (blue, DNA). #### File: BJ5ta_aSMA_BR1_Recovery.lif
 **Description:** BJ-5ta cells passed through 1round of 5 micron Transwell
 migration, allowed to proliferate for 2 days, then stained for smooth
 muscle alpha actin (488, green) and DAPI (blue, DNA). #### File:
 BJ5ta_5ngTGFb_aSMA.lif **Description:** BJ-5ta cells treated with 5 ng/mL
 TGFbeta for 48 hours, then stained for smooth muscle alpha actin (488,
 green) and DAPI (blue, DNA). ## Code/software .lif format stands for Leica
 Image Files and contain all z-stack images and include metadata for how
 images were acquired. Files can be opened using FIJI/ImageJ or Leica LASX
 Lite software for viewing and analysis. ## Access information Other
 publicly accessible locations of the data: * None Data was derived from
 the following sources: * None