Journal: Cell Communication and Signaling : CCS

Article Title: CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity

doi: 10.1186/1478-811X-11-40

Figure Lengend Snippet: Impaired binding of ZO-2 to the phospho-mimetic Occ-T400E/T404E/S408E construct. A ) The indicated GST-Occ cytoplasmic tail fusion proteins were used to pull down HA-ZO-2 from transiently transfected MDCK C11 cells with GSH-agarose beads. Isolated protein complexes were analyzed by Western blotting with anti-HA antibody. Equal amounts of GST- fusion proteins were pulled down as detected with an anti-GST antibody in the lower panel. B ) Quantification of 5 independent experiments as shown in (A). C ) Purified GST-occludin C-terminal domain (GST-OccC) was prephosphorylated in vitro by purified CK2 and subsequently used to pull down FLAG-tagged ZO-2 from transiently transfected HEK-293 cells. Association of FLAG-ZO-2 was analyzed by Western blotting with the anti-FLAG M2 antibody. D ) Densitometric quantification of 4 experiments as shown in ( C ).

Article Snippet: For immunofluorescence microscopy 1 × 10 6 MDCK C11 cells per well were seeded on chamber slides coated with collagen (0.1 μg/μl; Biochrom) and incubated for 24 h at 37°C, 5% CO 2 .

Techniques: Binding Assay, Construct, Transfection, Isolation, Western Blot, Purification, In Vitro

Journal: Cell Communication and Signaling : CCS

Article Title: CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity

doi: 10.1186/1478-811X-11-40

Figure Lengend Snippet: Localization of wildtype occludin-FLAG 3 and the corresponding T400/T404/S408 mutated constructs. MDCK C11 cells stably transfected with the indicated FLAG 3 -tagged occludin constructs were stained with anti-ZO-1 (red) and anti-FLAG M2 (green) antibodies and nuclei were stained with DAPI (clones: mock 3.1, wt 1.1, T400A/T404A/S408A 3.1, T400E/T404E/S408E 4.1). Representative images of the indicated clones are shown. Images were taken on a confocal laser-scanning microscope. The lower right panel represents a merged image of the other three images. Bar, 20 μM.

Article Snippet: For immunofluorescence microscopy 1 × 10 6 MDCK C11 cells per well were seeded on chamber slides coated with collagen (0.1 μg/μl; Biochrom) and incubated for 24 h at 37°C, 5% CO 2 .

Techniques: Construct, Stable Transfection, Transfection, Staining, Clone Assay, Laser-Scanning Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity

doi: 10.1186/1478-811X-11-40

Figure Lengend Snippet: Expression of TJ proteins in the stably transfected MDCK C11 cells. A ) Claudin-1, claudin-2, ZO-1 and ZO-2 expression is not affected by the stable transfection of the indicated occludin-FLAG 3 constructs as detected by Western blotting. β-Actin was used as a loading control. Analysis of two different clones for each construct is shown. B ) Transfection of the indicated occludin constructs does not affect cell proliferation as investigated by a XTT-assay. The graph summarizes the results of 4 experiments including two clones of each construct (mean values +/− SEM).

Article Snippet: For immunofluorescence microscopy 1 × 10 6 MDCK C11 cells per well were seeded on chamber slides coated with collagen (0.1 μg/μl; Biochrom) and incubated for 24 h at 37°C, 5% CO 2 .

Techniques: Expressing, Stable Transfection, Transfection, Construct, Western Blot, Control, Clone Assay, XTT Assay

Journal: Cell Communication and Signaling : CCS

Article Title: CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity

doi: 10.1186/1478-811X-11-40

Figure Lengend Snippet: Phosphorylation of occludin T400/T404/S408 regulates assembly/disassembly of TJs in Ca 2+ -switch experiments. A ) Confocal images were taken at t = 0 min after removal of Ca 2+ . B ) After depletion of Ca 2+ the phospho-mimetic Occ-T400E/T404E/S408E protein is rapidly dissociated from the TJs along with a loss of ZO-1 tight junctional staining. Wildtype occludin and Occ-T400A/T404A/S408A did not differ in the kinetics of disassembly. C ) After re-addition of Ca 2+ wildtype occludin and Occ-T400A/T404A/S408A rapidly reassembled into TJs whereas formation of TJs in Occ-T400E/T404E/S408E-transfected MDCK C11 cells was significantly delayed. Confocal images were taken 20 min after addition of Ca 2+ . Bar, 10 μM.

Article Snippet: For immunofluorescence microscopy 1 × 10 6 MDCK C11 cells per well were seeded on chamber slides coated with collagen (0.1 μg/μl; Biochrom) and incubated for 24 h at 37°C, 5% CO 2 .

Techniques: Phospho-proteomics, Staining, Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity

doi: 10.1186/1478-811X-11-40

Figure Lengend Snippet: Increase of paracellular resistance after CK2-dependent phosphorylation of occludin. Two-path impedance spectroscopy was applied to measure the two components of epithelial resistance (R epi ), paracellular resistance (R para , reflecting the pathway across the tight junctions) and transcellular resistance (R trans , reflecting the pathway across the cell membranes). Measurements were done in MDCK C11 cells, which were stably transfected with the indicated occludin-FLAG 3 constructs. In Occ-FLAG 3 -T400E/T404E/S408E-transfected cells a dramatic increase in R para was detectable compared to wildtype occludin and to Occ-FLAG 3 -T400A/T404A/S408A-transfected cells, while R trans was unchanged. Due to the about fourfold increase of R para , the overall epithelial resistance R epi was also increased. The figure shows combined results of 6 independent measurements on two clones of each construct. ** p < 0.01.

Article Snippet: For immunofluorescence microscopy 1 × 10 6 MDCK C11 cells per well were seeded on chamber slides coated with collagen (0.1 μg/μl; Biochrom) and incubated for 24 h at 37°C, 5% CO 2 .

Techniques: Phospho-proteomics, Impedance Spectroscopy, Stable Transfection, Transfection, Construct, Clone Assay

Journal: Viruses

Article Title: Insights into Human Astrocyte Response to H5N1 Infection by Microarray Analysis

doi: 10.3390/v7052618

Figure Lengend Snippet: HM/06 replicated efficiently in U251 cells and induced apoptosis in U251; ( A ) U251 cells were infected by HM/06 at MOI 1.0, and cells were lysed for NP detecting by western blot at indicated times post-infection; ( B ) HM/06-infected U251 cells were fixed, permeabilized, and stained with corresponding antibodies for immunofluorescence observation with an OLYMPUSIX70microscope. Bar, 50 µm; ( C ) U251 cells were infected by HM/06 at MOI 1.0, and at indicated times post-infection, supernatants were collected and viruses titer were determined by TCID 50 in MDCK cells. Data was showed as means ± SEM from three experiments; ( D ) U251 cells were infected by HM/06, and at 6, 12, and 24hpi, cells were washed with PBS, detached from the culture plates. Apoptosis was detected using FITC-Annexin V Apoptosis Detection Kit. Data were presentedas mean ± SEM from three experiments. Statistical significance was analyzed by student’s t -test. * p < 0.05, *** p < 0.001.

Article Snippet: Human astrocyte cells U251 and Madin-Darby canine kidney (MDCK) cells were obtained from China Center for Type Culture Collection (CTCC, Wuhan, China),maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, UT, USA), and incubated in 37 °C humidified incubator with 5% CO 2 .A/duck/Hubei/hangmei01/2006(H5N1) (HM/06) isolated from duck brain tissues[ ], which can cause neurovirulence and mortality in ducks, was propagated in nine-day-old specific pathogen-free embryonated eggs and titrated in MDCK cells by common plaque assays.

Techniques: Infection, Western Blot, Staining, Immunofluorescence

Journal: Scientific Reports

Article Title: TRPA1-dependent reversible opening of tight junction by natural compounds with an α,β-unsaturated moiety and capsaicin

doi: 10.1038/s41598-018-20526-7

Figure Lengend Snippet: Discovery of four natural compounds that increase TJ permeability reversibly by a Ca 2+ influx assay in the MDCK II cell monolayer. ( A ) The structures of capsaicin ( 1 ) and the screened compounds containing an α,β-unsaturated moiety identified in this study: pyrenocine A ( 2 ), pyrenocine H ( 3 ), dehydrocurvularin ( 4 ), and avenaciolide ( 5 ). α,β-unsaturated moiety is enclosed. The theoretical clog P -values of the four compounds are also shown. ( B ) Capsaicin induced a Ca 2+ influx in the MDCK II monolayer. ○: EtOH. □: 10 μM ionomycin. ●: 300 μM capsaicin. Typical data of three independent experiments is shown. ( C ) Four hit compounds induced a Ca 2+ influx in the MDCK II monolayer. ○: DMSO. □: 10 μM ionomycin. ●: 30 μM compound 2 . ■: 30 μM compound 3 . ▲: 10 μM compound 4 . ◆: 10 μM compound 5 . Typical data of three independent experiments is shown. ( D ) Capsaicin reversibly increased the paracellular permeability in the MDCK II monolayers, whereas LatA irreversibly increased it. ○: EtOH. □: 0.1 μM LatA. ●: 300 μM capsaicin. Typical data of three independent experiments is shown. ( E ) The four hit compounds reversibly increased the paracellular permeability in the MDCK II monolayer. ○: DMSO. ●: 30 μM compound 2 . ■: 30 μM compound 3 . ▲: 10 μM compound 4 . ◆: 10 μM compound 5 . Typical data of three independent experiments is shown. ( F ) The efficiencies of TJ permeability enhancement induced by the compounds were evaluated by the cumulative FD4 amounts transported until 2 h relative to vehicle control. Each value represents the mean ± S.D. of three independent experiments. ( G ) Reversibilities of TJ permeability induced by the compounds were evaluated by reversibility indexes (RIs). The results of ≥3 independent experiments were used to calculate each compound’s RI. p -values were calculated by Tukey’s multiple comparison test.

Article Snippet: Madin-Darby canine kidney (MDCK) II cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal calf serum (Nichirei Biosciences, Tokyo) and 1% penicillin-streptomycin (Nacalai Tesque) in a humidified atmosphere containing 5% CO 2 .

Techniques: Permeability, Control, Comparison

Journal: Scientific Reports

Article Title: TRPA1-dependent reversible opening of tight junction by natural compounds with an α,β-unsaturated moiety and capsaicin

doi: 10.1038/s41598-018-20526-7

Figure Lengend Snippet: SAR analysis of four natural α,β-unsaturated compounds with TJ opening ability. ( A ) The structures of compounds 6 – 9 and NEM are shown. The compounds 6 and 7 are analogs of compounds 2 and 3 . Compounds 8 and 9 are analogs of compound 4 and compound 5 , respectively. α,β-unsaturated moiety of compounds 9 and NEM is enclosed. The theoretical clog P- values of compounds 6 – 9 are also shown. ( B ) The structural analogs that have no α,β-unsaturated carbonyl group: compounds 6 and 7 did not increase paracellular permeability in MDCK II monolayers. ○: DMSO. □: 0.1 μM LatA: ●: 30 μM compound 2 ■: 30 μM compound 6 . ▲: 30 μM compound 7 . Typical data of three independent experiments is shown. ( C ) The structural analog of compound 5 : compound 9 did not increase paracellular permeability in MDCK II monolayers, although it has an α,β-unsaturated carbonyl group. ○: DMSO. □: 0.1 μM LatA. ●: 10 μM compound 5 . ■: 10 μM compound 9 . Typical data of three independent experiments is shown.

Article Snippet: Madin-Darby canine kidney (MDCK) II cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal calf serum (Nichirei Biosciences, Tokyo) and 1% penicillin-streptomycin (Nacalai Tesque) in a humidified atmosphere containing 5% CO 2 .

Techniques: Permeability

Journal: Scientific Reports

Article Title: TRPA1-dependent reversible opening of tight junction by natural compounds with an α,β-unsaturated moiety and capsaicin

doi: 10.1038/s41598-018-20526-7

Figure Lengend Snippet: Involvement of the α,β-unsaturated moiety as a potent pharmacophore to open TJs. ( A ) NEM induced Ca 2+ influx in MDCK II monolayers. ○: DMSO. □: 0.1 μM LatA. ●: 30 μM NEM. Typical data of three independent experiments is shown. ( B ) NEM increased the paracellular permeability in the MDCK II monolayer, but DTT pretreatment for 10 min abolished the permeability increase. ○: DMSO, □: 0.1 μM LatA, ●: 30 μM NEM, and ■: 30 μM NEM pretreated with 30 μM DTT. Typical data of three independent experiments is shown. ( C ) DTT pretreatment for 10 min did not affect the capsaicin-induced TJ opening. ○: EtOH, □: 0.1 μM LatA, ●: 300 μM capsaicin, and ■: 300 μM capsaicin pretreated with 300 μM DTT. Typical data of three independent experiments is shown. ( D ) DTT pretreatment abolished the permeability increase induced by compound 2 . ○: DMSO, □: 0.1 μM Lat A, ●: 30 μM compound 2 , and ■: 30 μM compound 2 pretreated with 30 μM DTT. Typical data of five independent experiments is shown.

Article Snippet: Madin-Darby canine kidney (MDCK) II cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal calf serum (Nichirei Biosciences, Tokyo) and 1% penicillin-streptomycin (Nacalai Tesque) in a humidified atmosphere containing 5% CO 2 .

Techniques: Permeability

Journal: Scientific Reports

Article Title: TRPA1-dependent reversible opening of tight junction by natural compounds with an α,β-unsaturated moiety and capsaicin

doi: 10.1038/s41598-018-20526-7

Figure Lengend Snippet: Mechanistic similarities/differences underlying the reversible opening of TJ between capsaicin and α,β-unsaturated compounds. ( A ) Western blot detection of cofilin phosphorylation, occludin and claudin-1. MDCK II monolayers were treated with ( left panel ) 300 μM capsaicin and 30 μM compound 2 , and ( right panel ) 300 μM capsaicin, 10 μM compounds 4 and 5 for indicated durations. Typical data of three independent experiments is shown. The samples of upper and lower panels derived from the same experiment and full length figures are presented in Suppl Fig. . ( B ) The densitometric analysis of phosphorylated cofilin ( left ) and occludin ( right ) from three independent experiments performed with NIH ImageJ software. The band intensity of each time point relative to that of time 0 are shown. Student’s t-test for compound 2 and Dunnett’s test for compounds 4 and 5 has been performed comparing each compound with capsaicin at each time point. ( C ) Compound 2 and 4 affected the distribution of actin filament similarly to capsaicin, but NEM and compound 5 did not. MDCK II monolayers were treated with 300 μM capsaicin, 30 μM compound 2 and NEM, 10 μM compounds 4 and 5 for 45 min. Scale bar: 10 μm. Typical data of three independent experiments is shown.

Article Snippet: Madin-Darby canine kidney (MDCK) II cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal calf serum (Nichirei Biosciences, Tokyo) and 1% penicillin-streptomycin (Nacalai Tesque) in a humidified atmosphere containing 5% CO 2 .

Techniques: Western Blot, Phospho-proteomics, Derivative Assay, Software

Journal: Scientific Reports

Article Title: TRPA1-dependent reversible opening of tight junction by natural compounds with an α,β-unsaturated moiety and capsaicin

doi: 10.1038/s41598-018-20526-7

Figure Lengend Snippet: Compound 2 and capsaicin both induced a Ca 2+ influx and a permeability increase via activation of TRPA1 in MDCK II monolayers. ( A ) TRPA1 antagonist A-967079 pretreatment for 30 min abolished the Ca 2+ influx induced by compound 2 in the MDCK II monolayer. Typical data of three independent experiments is shown. ○: DMSO, □: 10 μM ionomycin, △: 30 μM compound 2 , ▲: 30 μM compound 2 pretreated with 1 μM A-967079, and ●: 1 μM A-967079. ( B ) TRPA1 antagonist A-967079 pretreatment for 30 min inhibited the permeability increase induced by compound 2 in MDCK II monolayer. Typical data of three independent experiments is shown. ○: DMSO, △: 30 μM compound 2 , ▲: 30 μM compound 2 pretreated with 1 μM A-967079, and ●: 1 μM A-967079. ( C ) TRPA1 antagonist A-967079 pretreatment for 30 min abolished the capsaicin-induced Ca 2+ influx in the MDCK II monolayer. Typical data of three independent experiments is shown. ○: DMSO, □: 10 μM ionomycin, △: 300 μM capsaicin, ▲: 300 μM capsaicin pretreated with 1 μM A-967079, and ●: 1 μM A-967079. ( D ) TRPA1 antagonist A-967079 pretreatment for 30 min inhibited the capsaicin-induced permeability increase in the MDCK II monolayer. Typical data of three independent experiments is shown. ○: DMSO, △: 300 μM capsaicin, ▲: 300 μM capsaicin pretreated with 1 μM A-967079, ●: 1 μM A-967079. ( E ) Cofilin dephosphorylation induced by 300 μM capsaicin was inhibited by pretreatment with 1 μM A-967079 for 30 min. Typical data of three independent experiments is shown. The samples derived from the same experiment and full length figures are presented in Suppl. Fig. .

Article Snippet: Madin-Darby canine kidney (MDCK) II cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal calf serum (Nichirei Biosciences, Tokyo) and 1% penicillin-streptomycin (Nacalai Tesque) in a humidified atmosphere containing 5% CO 2 .

Techniques: Permeability, Activation Assay, De-Phosphorylation Assay, Derivative Assay

Journal: Scientific Reports

Article Title: TRPA1-dependent reversible opening of tight junction by natural compounds with an α,β-unsaturated moiety and capsaicin

doi: 10.1038/s41598-018-20526-7

Figure Lengend Snippet: TRPA1 agonist AITC, compound 2 and capsaicin failed to induce TJ permeability increase in TRPA1-KO MDCK II monolayers. ( A ) TRPA1 agonist AITC induced a Ca 2+ influx in the MDCK II monolayer, as did compound 2 and capsaicin. Typical data of three independent experiments is shown. ○: DMSO, □: 10 μM ionomycin, △: 30 μM AITC. ■: 30 μM compound 2 . ●: 300 μM capsaicin. Typical data of three independent experiments is shown. ( B ) TRPA1 agonist AITC failed to induce a Ca 2+ influx in the TRPA1-KO MDCK II monolayer. Typical data of three independent experiments is shown. ○: DMSO, □: 10 μM ionomycin, △: 30 μM AITC. ■. 30 μM compound 2 . ●: 300 μM capsaicin. ( C ) TRPA1 agonist AITC increased the TJ permeability in the MDCK II monolayer but not in the TRPA1-KO monolayer. Typical data of three independent experiments is shown. Upper graph : WT MDCK II monolayer. Lower graph : TRPA1-KO MDCK II monolayer. ○: DMSO. □: 0.1 μM Lat A. △: 30 μM AITC. ( D ) The TJ permeability increases induced by both compound 2 and capsaicin in the MDCK II monolayer were abolished in the TRPA1-KO monolayer. Typical data of three independent experiments is shown. Upper graph : WT MDCK II monolayer. Lower graph : TRPA1-KO MDCK II monolayer. ○: DMSO. □: 0.1 μM Lat A. ■: 30 μM compound 2 . ●: 300 μM capsaicin. ( E ) Cofilin dephosphorylation was induced by 300 μM capsaicin in the WT MDCK II monolayer but not in the TRPA1-KO monolayer. The samples derived from the same experiment and full length figures are presented in Suppl Fig. .

Article Snippet: Madin-Darby canine kidney (MDCK) II cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal calf serum (Nichirei Biosciences, Tokyo) and 1% penicillin-streptomycin (Nacalai Tesque) in a humidified atmosphere containing 5% CO 2 .

Techniques: Permeability, De-Phosphorylation 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 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