cell lines mdck ii  (ATCC)

Journal: Communications Biology

Article Title: Regulation of chromatin modifications through coordination of nucleus size and epithelial cell morphology heterogeneity

doi: 10.1038/s42003-025-07677-w

Figure Lengend Snippet: A Images of MDCK cells demonstrating evolution in cell and nucleus morphology from being subconfluent to crowded (top). Cross-sectional 3D reconstruction of MDCK cells demonstrating monolayer flatness throughout crowding (middle). Scale bar = 10 μm. DAPI and actin staining shows that cells progressively acquire a cobblestone-like morphology with decreasing sizes of both cell and nucleus during crowding. E-cadherin (E-cad) staining illustrates the maturation of intercellular junctions in late crowding. Scale bar = 50 μm. B Quantification of cell area throughout crowding illustrates that the cell area variability surpasses the mean change. N = 124, 732, 803, and 1033 for 24, 64, 72, and 104 h analyses, respectively. C Quantification of nucleus area throughout crowding illustrates that the nucleus area variability also surpasses the mean change. N = 147, 837, 840, and 1107 for 24, 64, 72, and 104 h analyses, respectively. D Persisting nucleus-cell area correlation throughout crowding. 64 h, 72 h, 104 h datasets exhibit the same NC ratio, indicated by the same slope of the best fits (solid lines). N = 124, 808, 802, and 1033 for 24, 64, 72, and 104 h analyses, respectively. Solid lines represent best linear fits. p < 0.0001 for all timepoints. 95% confidence intervals corresponding to the 24, 64, 72, and 104 h data are [0.518, 0.731], [0.810, 0.853], [0.771, 0.822], and [0.668, 0.731], respectively. Correlation coefficients corresponding to the 24, 64, 72, and 104 h data are 0.637, 0.804, 0.751, and 0.721, respectively. E Normalized probability density functions (PDF) for MDCK, HaCaT, and developing E12.5 mouse epithelium cell and nucleus area. All PDFs, except the MDCK cell 24 h PDF, collapse on a master curve and can be described by a log-normal fit. F Quantification of cell aspect ratio (AR) throughout crowding. N = 124, 732, 803, and 1033 for 24, 64, 72, and 104 h analyses, respectively. G Quantification of nucleus AR throughout crowding. N = 124, 808, 803, and 1033 for 24, 64, 72, and 104 h analyses, respectively. H Nucleus-cell AR correlation during crowding showing progressively increased slopes over time. Solid lines represent best linear fits (solid lines). N = 124, 808, 803, and 1033 for 24, 64, 72, and 104 h analyses, respectively. p < 0.0001 for all timepoints. 95% confidence intervals corresponding to the 24, 64, 72, and 104 h data are [0.134, 0.455], [0.366, 0.479], [0.388, 0.499], and [0.533, 0.614], respectively. Correlation coefficients corresponding to the 24, 64, 72, and 104 h data are 0.303, 0.424, 0.386, and 0.575, respectively. I Normalized PDFs for MDCK, HaCaT, and mouse epithelium cell and nucleus AR collapse on a master curve, which can be described by a gamma distribution. **** refers to p < 0.0001. 3 biological replicates were used for all analyses.

Article Snippet: All experiments conducted using Madin Darby Canine Kidney cells (MDCK II cell line) were cultured in MEM- α (Fisher Scientific, 12561-056) supplemented with 10% fetal bovine serum (FBS) (Fisher Scientific, 12662-029) and 1% Penicillin-Streptomycin (Fisher Scientific, 15140-122).

Techniques: Staining

Journal: Communications Biology

Article Title: Regulation of chromatin modifications through coordination of nucleus size and epithelial cell morphology heterogeneity

doi: 10.1038/s42003-025-07677-w

Figure Lengend Snippet: A Image of MDCK cells stained with Histone H3 Lysine 27 trimethylation (H3K27me3) illustrating its variable expression levels in different cells. Scale bar = 50 μm. B Correlation between normalized H3K27me3 expression level and nucleus area in MDCK cells. N = 461. 95% confidence interval is [−0.447, −0.194]. C Image of an E12.5 mouse epithelium stained with H3K27me3. Scale bar = 25 μm. D Correlation between normalized H3K27me3 expression and nucleus area in the mouse epithelium. N = 130. 95% confidence interval is [−0.459, −0.209]. E Image of MDCK cells stained with Histone H3 Lysine 9 acetylation (H3K9ac). Scale bar = 50 μm. F Correlation between normalized H3K9ac expression and nucleus area in MDCK cells. N = 1130. 95% confidence interval is [0.183, 0.293]. G Image of an E12.5 mouse epithelium stained with H3K9ac. Scale bar = 25 μm. H Correlation between normalized H3K9ac expression and nucleus area in the mouse epithelium. N = 713. 95% confidence interval is [0.0988, 0.241]. I Schematic of the nucleus region split for radial distribution analysis. Yellow shaded region occupying 80% of total nucleus area is classified as the center, while the outer 20% shaded in blue is classified as the periphery. J Airyscan image of a small (top) and large (bottom) nucleus of crowded MDCK cells illustrating differences in the radial distribution of H3K9ac. Scale bar = 5 μm. K Correlation between H3K9ac periphery-center ratio and nucleus area of crowded MDCK cells. N = 1246. 95% confidence interval is [0.131, 0.238]. L Summary of the absolute value of the Pearson correlation coefficient between nucleus area and intensity or spatial distribution analyses in crowded MDCK cells. M Images of unconfined (left) and confined (right) MDCK cells stained with H3K27me3 (top) or H3K9ac (bottom). Cells were confined using circular 10 μm fibronectin micro-patterned substrates. Scale bar = 50 μm. N Box and whisker chart demonstrating an increase of normalized H3K27me3 intensity in confined cells. Data were obtained from N = 59 and 75 nuclei derived from three independent stamp samples for unconfined and confined cells, respectively. O Box and whisker chart showing a decrease of normalized H3K9ac intensity in confined cells. N = 50 and 52 nuclei derived from three independent stamp samples for unconfined and confined cells, respectively. *, **, and ***, refer to p < 0.05, <0.01, and <0.001 respectively. N = 3 biological replicates were used for all plots.

Article Snippet: All experiments conducted using Madin Darby Canine Kidney cells (MDCK II cell line) were cultured in MEM- α (Fisher Scientific, 12561-056) supplemented with 10% fetal bovine serum (FBS) (Fisher Scientific, 12662-029) and 1% Penicillin-Streptomycin (Fisher Scientific, 15140-122).

Techniques: Staining, Expressing, Whisker Assay, Derivative Assay

Journal: Communications Biology

Article Title: Regulation of chromatin modifications through coordination of nucleus size and epithelial cell morphology heterogeneity

doi: 10.1038/s42003-025-07677-w

Figure Lengend Snippet: A Schematic illustrating cytoplasmic and nuclear features used in morphological analyses. CV refers to coefficient of variation of DAPI intensity. B Prediction-measurement correlation with nucleus area. Axis range has been optimized to highlight differences between groups. Group titled “Cell Morph” refers to cell morphology illustrated in ( A ). GPR and CCA denote Gaussian process regression and canonical correlation analysis, respectively. C Principal component analysis biplot of cell morphology, nuclear morphology, and H3K27me3 levels. PC indicates principal component. D Prediction-measurement correlation for H3K27me3 levels. Groups titled “Cell Morph” and “Nuc Morph” refer to morphologies illustrated in ( A ). Nonlinearity group utilizes GPR model. E H3K27me3 level predicted using GPR versus measured level. Gray shaded band represents the GPR 95% confidence interval. Red dashed line denotes a perfect correlation ( r = 1). N = 1080. F Illustration of measured H3K27me3 levels in MDCK cells (top) and predicted levels using GPR (bottom). Color bar indicates normalized H3K27me3 level. G H3K27me3 prediction-measurement correlation for control and DN-KASH (KASH) using nucleus area as the sole predictor. H H3K27me3 prediction-measurement correlation obtained using multi-linear regression. I H3K27me3 prediction-measurement obtained using GPR. J Predictor importance rank comparison scatter plot between control and KASH cells. Key morphological features for control and KASH samples are emphasized in fuchsia and cyan, respectively. Correlation measurements were conducted using N = 376, N = 335, and N = 446 cells for ( B , D ); N = 1277, N = 1346, and N = 1101 cells for the control condition in ( G – I ); and N = 1262, N = 1438, and N = 1147 cells for the KASH condition in ( G – I ). “ns'', *, **, ***, ****, refer to p ≥ 0.05, < 0.05, < 0.01, < 0.001, and < 0.0001, respectively. 3 biological replicates were used for ( B – F ), while 4 biological replicates were used for ( G – J ).

Article Snippet: All experiments conducted using Madin Darby Canine Kidney cells (MDCK II cell line) were cultured in MEM- α (Fisher Scientific, 12561-056) supplemented with 10% fetal bovine serum (FBS) (Fisher Scientific, 12662-029) and 1% Penicillin-Streptomycin (Fisher Scientific, 15140-122).

Techniques: Control, Comparison

Journal: Communications Biology

Article Title: Regulation of chromatin modifications through coordination of nucleus size and epithelial cell morphology heterogeneity

doi: 10.1038/s42003-025-07677-w

Figure Lengend Snippet: A MDCK cells stained for UTX with example images of small (upper right) and large nuclei (bottom right). B The UTX/DAPI intensity of the smallest 20% of nuclei (Small) is significantly lower than that of largest 20% of nuclei (Large). N = 295 pooled from 3 biological replicates. C MDCK cells stained for EZH2 with example images of small (upper right) and large nuclei (bottom right). D EZH2 levels displayed no significant difference between small and large nuclei. N = 295 pooled from 3 biological replicates. E EZH2/UTX intensity ratio is higher in small nuclei than in large nuclei. N = 295 pooled from 3 replicates. F MDCK cells stained for H3K27me3 in control, GSK-J1 and DS3201 samples. G GSK-J1 and DS3201 respectively increased and decreased the H3K27me3 levels. N = 77, 217, and 294 for control, GSK-J1, and DS3201, respectively. H Pearson correlation coefficient between nucleus size and H3K27me3 intensity for control, GSK-J1, and DS3201 treated cells. GSK-J1 and DS3201 treatments reduced the anti-correlation between nucleus size and H3K27me3 intensity. Dotted line denotes no correlation. I MDCK cells stained for UTX in control and blebbistatin-treated (Bleb) samples. J Bleb samples exhibited lower UTX levels than control. N = 6. K Pearson correlation coefficient between nucleus size and H3K27me3 intensity for control and Bleb samples. Dotted line denotes no correlation. L Fluorescent images illustrating UTX levels in unconfined (top) and 10 μm-confined (bottom) cells. White dashed outline in confined cells denotes nuclear contour. M Quantification of UTX intensity normalized to DAPI intensity in unconfined and confined cells. N Same as ( L ) but for H3K27me3. O Same as ( M ) but for H3K27me3. P Proposed mechanism of how heterogeneous cell morphology generates diversity in nucleus size and chromatin states. In a crowded epithelial monolayer, a mother cell divides unevenly into two daughter cells with different sizes, which persist thereafter. Each cell size then propagates through actomyosin tension and intracellular osmotic pressure balance to determine the corresponding nucleus size. Cell size heterogeneity thus gives rise to nucleus size heterogeneity, which in turn contributes to the varying UTX levels and chromatin modifications. Scale bar = 10 μm for ( A , C , F , I ). Scale bar = 20 μm for ( L , N ). p < 0.05 for ( B , D , F , H , K ). “ns'', *, **, ***, ****, refer to p ≥ 0.05, <0.05, <0.01, <0.001, and <0.0001, respectively. 3 biological replicates were used for all analyses.

Article Snippet: All experiments conducted using Madin Darby Canine Kidney cells (MDCK II cell line) were cultured in MEM- α (Fisher Scientific, 12561-056) supplemented with 10% fetal bovine serum (FBS) (Fisher Scientific, 12662-029) and 1% Penicillin-Streptomycin (Fisher Scientific, 15140-122).

Techniques: Staining, Control