htert rpe 1 cells  (ATCC)

Journal: iScience

Article Title: CTCF/cohesin-binding sites are susceptible to replication-associated DNA damage and genomic instability in cancer cells

doi: 10.1016/j.isci.2026.114646

Figure Lengend Snippet: Replication stress and DNA damage response at CTCF/cohesin-binding sites in normal cells (A–D) ChIP-seq signal profile and heatmap of (A) MRE11, (B) STN1+HU 3h, (C) γH2AX (normalized with H2AX), and (D) RAD51 in the Mid S phase of HeLa cells plotted at CBSs shared between HeLa and normal cells (H1, IMR90, and RPE1 cells) (12,517 sites) and HeLa-specific CBSs (2,293 sites) and normal-specific sites (13,702). For the γH2AX signal ±10 kb flanks were considered, while for others the signal is plotted at ±5 kb regions. (E–H) ChIP-qPCR plots of (E) MRE11, (F) FANCD2, (G) γH2AX, and (H) RAD51 in Mid S synchronized hTERT RPE-1 cells at CBSs and CTCF-unbound sites. The y axis (fold enrichment over beads) indicates the % input in immunoprecipitation divided by that of beads. The bar represents the mean value from three replicates, and the error bar represents the standard error of the mean. Statistical significance was determined by using a two-sided Mann-Whitney U test.

Article Snippet: HeLa cells (obtained from ATCC) were grown in High Glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, ThermoFisher, #10569044), supplemented with 10% Fetal Bovine Serum (FBS, Gibco, Invitrogen #16000044) and 1% penicillin-streptomycin (Gibco, Invitrogen #15140163). hTERT RPE-1 cells (purchased from ATCC, #CRL-4000) were cultured in DMEM/F12 (Gibco, Thermofisher #10565042) with 10% FBS and 1% penicillin-streptomycin.

Techniques: Binding Assay, ChIP-sequencing, ChIP-qPCR, Immunoprecipitation, MANN-WHITNEY

Journal: bioRxiv

Article Title: A gene expression correlation analysis of cell mechanosensitive systems identifies DANGER as a nuclear mechanoresponsive protein

doi: 10.64898/2026.01.26.701746

Figure Lengend Snippet: a) Strategy map to identify candidate genes involved in mechanotransduction pathways. b) Heatmap of the proteins encoded by the MC genes (divided into the three signatures), ranked based on their z-score of Pearson correlation, and their presence in the different cell organelles/structures or compartments. c) Percentage of the proteins of each organelle/structure/compartment present in each of the indicated signatures. d, e) List of MC genes -separated by signatures-that are present in the nuclear envelope (d) or mitochondria (e) proteome. Proteins are ranked based on their presence in the different signatures and based on their z-score of Pearson correlation. f) Shared genes in the three signatures. g) Immunofluorescence of RPE1 cells stained for the indicated proteins and the nucleus. Cells were treated with hypo-osmotic shock and control media for 10 minutes before fixation. Scale bar 10 μm.

Article Snippet: RPE1 cells (ATCC CRL-4000) were grown in DMEM/F-12 (Lonza).

Techniques: Immunofluorescence, Staining, Control

Journal: bioRxiv

Article Title: A gene expression correlation analysis of cell mechanosensitive systems identifies DANGER as a nuclear mechanoresponsive protein

doi: 10.64898/2026.01.26.701746

Figure Lengend Snippet: a) Immunofluorescence of RPE1 cells stained for DANGER and the nucleus. Cells were treated with the indicated siRNAs, fixed and stained. Scale bar 20 and 10 (inset) μm. b) Western blot of the indicated antibodies on RPE1 cell lysates transfected with the indicated siRNAs. c) Quantification of the DANGER signal in the nucleus in control and DANGER silenced RPE1 cells. Data represent mean ± s.e.m. N = 58 cells for each condition, from 3 independent experiments. d) Immunofluorescence of the indicated cells stained for the DANGER and the nucleus. Scale bar 10 μm. e) Immunofluorescence of RPE1 cells stained for the indicated proteins and the nucleus. Confocal sections were taken in the Y plane from the whole nuclear section and the maximum projection is shown. Scale bar 5 μm. f) DANGER spots are organized into heterogenous populations. Histogram of DANGER spots segmented and quantified. The frequency of each spot area range is represented. N = 1021 spots representative of 4 independent experiments.

Article Snippet: RPE1 cells (ATCC CRL-4000) were grown in DMEM/F-12 (Lonza).

Techniques: Immunofluorescence, Staining, Western Blot, Transfection, Control

Journal: bioRxiv

Article Title: A gene expression correlation analysis of cell mechanosensitive systems identifies DANGER as a nuclear mechanoresponsive protein

doi: 10.64898/2026.01.26.701746

Figure Lengend Snippet: a) Immunofluorescence of RPE1 cells stained for the indicated proteins and the nucleus. Confocal sections are shown on nuclei with blebs. The bleb is marked with an arrow. Scale bar 5 μm. b) Diagram of the three regions quantified in each nuclei containing blebs. c) Quantification of DANGER enrichment in nuclear blebs form panel a. Emerin and DNA signal was also quantified in the same regions. d) Immunofluorescence of PANC1 cells stained for the indicated proteins and the nucleus. Confocal sections are shown on multilobed nuclei with bents and bridges (marked with an arrow). Scale bar 10 μm. e) Quantification of DANGER enrichment in nuclear bridges from panel d. Emerin and DNA signal was also quantified in the same regions.

Article Snippet: RPE1 cells (ATCC CRL-4000) were grown in DMEM/F-12 (Lonza).

Techniques: Immunofluorescence, Staining

Journal: bioRxiv

Article Title: A gene expression correlation analysis of cell mechanosensitive systems identifies DANGER as a nuclear mechanoresponsive protein

doi: 10.64898/2026.01.26.701746

Figure Lengend Snippet: a) Immunofluorescence of RPE1 cells stained for the indicated proteins and the nucleus after treatment with iso-osmotic or hypo-osmotic medium for 10 minutes. Scale bar 20 μm. b) Quantification on DANGER spots in iso-osmotic (iso) and hypo-osmotic (Hypo) conditions. Box-and-whisker plots of spot intensity for both conditions with individual observations is shown. The box shows the IQR (25th–75th percentiles). Mann–Whitney U test p-value (two-sided) = 1.25 × 10 −17 . c) Quantification on DANGER spots intensity under iso-osmotic (iso) and hypo-osmotic (Hypo) conditions. Control spot populations (iso-osmotic) were divided into four quartiles based on spot intensity: q1 (low), q2 (low–medium), q3 (high–medium) and q4 (high). To compare proportions, a proportions test based on a χ -test was used. d) Immunofluorescence of U2OS cells stained for the indicated proteins and the nucleus after spreading on a fibronectin-coated surface for 2 (early spreading) and 6 hours (late spreading). Scale bar 10 μm. e) Quantification on DANGER spots in cells spread for 2 hours (early spreading) and 6 hours (late spreading). Box-and-whisker plots of spot intensity for both conditions with individual observations is shown. The box shows the IQR (25th–75th percentiles). Mann–Whitney U test p-value (two-sided) = 1.43 × 10 −12. . f) Quantification on DANGER spots intensity in cells spread for 2 hours (early spreading) and 6 hours (late spreading). Control spot populations (iso-osmotic) were divided into four quartiles based on spot intensity: q1 (low), q2 (low–medium), q3 (high–medium) and q4 (high). To compare proportions, a proportions test based on a χ -test was used.

Article Snippet: RPE1 cells (ATCC CRL-4000) were grown in DMEM/F-12 (Lonza).

Techniques: Immunofluorescence, Staining, Whisker Assay, MANN-WHITNEY, Control

Journal: bioRxiv

Article Title: A gene expression correlation analysis of cell mechanosensitive systems identifies DANGER as a nuclear mechanoresponsive protein

doi: 10.64898/2026.01.26.701746

Figure Lengend Snippet: a, b, c) RPE1 cells were transfected with siRNA control (siControl), DANGER (siDANGER). siDANGER siRNA was also transfected in cells expressing siRNA insensitive DANGER cDNA (siDANGER + DANGER Res). Cells were processed for immunofluorescence, stained with Hoechst and DANGER. The indicated nuclear morphometric parameters were quantified with imageJ (b and c). The image representing overexpressed DANGER (DANGER Res) was acquired with less intensity to avoid saturation; to compare levels in this condition with respect to the endogenous see western blot below (panel d). Data represent mean ± s.e.m. Data representative of 3 independent experiments. d) Western blot showing DANGER protein levels in RPE1 cells transfected with siControl or siDANGER and in RPE1 cells expressing DANGER siRNA insensitive DANGER. A dotted line indicates the films were cut and fused. e, f) U2OS cells were transfected with siRNA control (siControl), DANGER (siDANGER). siDANGER siRNA was also transfected in U2OS cells expressing siRNA insensitive DANGER cDNA (siDANGER + DANGER Res). Cells were processed for immunofluorescence, stained with Hoechst and quantified (e) for bleb frequency in nuclei. Data represent mean ± s.e.m. N = 3040 (siControl), N=2630 (siDANGER) and N=2614 (siDANGER + DANGER Res) from 3 independent experiments. Asterisks mark two cells not re-expressing DANGER, which consistently presented nuclear deformations.

Article Snippet: RPE1 cells (ATCC CRL-4000) were grown in DMEM/F-12 (Lonza).

Techniques: Transfection, Control, Expressing, Immunofluorescence, Staining, Western Blot