Journal: Nature materials

Article Title: Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers

doi: 10.1038/s41563-021-00919-2

Figure Lengend Snippet: a) Top, left and right: typical examples of traction force magnitude maps for a single MDCK WT and E-cadherin KO cell cultured on deformable PDMS surfaces. Bottom, left and right: vectorial maps of traction forces for a single MDCK WT and E-cadherin KO cell on a soft PDMS substrate. Scale bars, 20μm . b) Schematic showing the defect movement based on force balance for an extensile active nematic system (left) and contractile active nematic system (right) with an inset of forces exerted on neighbours by an extensile (left) and contractile (right) nematic particle. c) Schematic (left) and experimental (right) images of +1/2 defect (left, comet configuration) and - 1/2 defect (right, trefoil configuration). Scale bars, 20μm . d) Average yy - and xy components of strain rate map around + 1/2 defect obtained from experiments (left and middle respectively) and corresponding average flow field (right) for MDCK WT cells (top) ( n = 1934 defects from 2 independent experiments) and MDCK E-cadherin KO cells (bottom) ( n = 1,884 defects from 2 independent experiments). Schematic on the extreme right illustrates the movement of defects. Colour code is positive for stretching and negative for shrinkage. e, f) Experimental data for MDCK WT (e) and MDCK E-cadherin KO (f) monolayers. Top panels: phase contrast images of the cells overlaid with the average local orientation of the cells (red lines). Bottom panels: average local orientation of the cells (red lines). The blue circle shows the location of a +1/2 defect and the corresponding arrow indicates the direction of motion of this defect over time. Dashed lines have been added for better reading of defect movement. Scale bars, 40μm .

Article Snippet: Primary antibodies were diluted in PBS Tween at E-cadherin (24E10-1:1000 for MDCK cells) (Santa Cruz, SC7870-1:200 for MCF7 cells), α-catenin (1:1000), β -catenin (1:1000), vinculin (gift from Marina Glukhova, 1:500), GAPDH (Protein Tech Europe 60004-1 for MDCK cells and abcam- 1:5000, ab181603 for MCF7 cells- 1:500), alpha-tubulin (1:5000) (Sigma T9026) overnight on a shaker at 4 °C.

Techniques: Cell Culture

Journal: Nature materials

Article Title: Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers

doi: 10.1038/s41563-021-00919-2

Figure Lengend Snippet: a, b) Orientation field (left) and velocity vectors (right) around a single comet shaped (+1/2) defect (a) and trefoil (-1/2) defect obtained from WT (top) and E-cadherin KO (bottom) monolayers. c, d) Trajectory of several comet (+1/2) (left) and trefoil (-1/2) (right) shaped defects obtained from MDCK WT (c) and MDCK E-cadherin KO (d) monolayers. Scale bars: 40μm.

Article Snippet: Primary antibodies were diluted in PBS Tween at E-cadherin (24E10-1:1000 for MDCK cells) (Santa Cruz, SC7870-1:200 for MCF7 cells), α-catenin (1:1000), β -catenin (1:1000), vinculin (gift from Marina Glukhova, 1:500), GAPDH (Protein Tech Europe 60004-1 for MDCK cells and abcam- 1:5000, ab181603 for MCF7 cells- 1:500), alpha-tubulin (1:5000) (Sigma T9026) overnight on a shaker at 4 °C.

Techniques:

Journal: Nature materials

Article Title: Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers

doi: 10.1038/s41563-021-00919-2

Figure Lengend Snippet: a) Average isotropic stress around a +1/2 defect obtained from simulations for the control condition (left) and condition without intercellular forces (right) (n = 2,083 defects). b,c) Average yy (left)-, xy (middle)- and isotropic (right) components of stress around a + 1/2 defect obtained from experiments for (b) MDCK WT (n = 1,899 defects) and (c) E-cadherin KO (n = 1,428 defects) from 2 independent experiments. For a and b colour code represents the strength of the stress with positive for tensile state, negative for compression. d, e, f) velocity correlation length (d) (n=10), velocity (e) (n=10) and mean traction force (f) (n=12) of cells within a monolayer for both MDCK WT and MDCK E-cadherin KO cells. g, h) Cell spreading area (g) and aspect ratio (h) of cells within the monolayer obtained from n=10 different images for MDCK WT and E-cadherin KO cells as a function of time from 2 independent experiments. The error bars represent the standard deviation. Unpaired t-test was performed resulting in *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Article Snippet: Primary antibodies were diluted in PBS Tween at E-cadherin (24E10-1:1000 for MDCK cells) (Santa Cruz, SC7870-1:200 for MCF7 cells), α-catenin (1:1000), β -catenin (1:1000), vinculin (gift from Marina Glukhova, 1:500), GAPDH (Protein Tech Europe 60004-1 for MDCK cells and abcam- 1:5000, ab181603 for MCF7 cells- 1:500), alpha-tubulin (1:5000) (Sigma T9026) overnight on a shaker at 4 °C.

Techniques: Control, Standard Deviation

Journal: Nature materials

Article Title: Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers

doi: 10.1038/s41563-021-00919-2

Figure Lengend Snippet: Immunofluorescence staining (top) of E-cadherin (left), β-catenin (middle) and ZO1 (right), along with a b) representative western blot and quantification for E-cadherin (left) (n=3) and β-catenin (right) (n=3). Scale bars, 20μm. c) Western blot analysis of total MLC and quantification from 3 independent experiments normalized to α-tubulin. d) Western blot analysis of vinculin and quantification from 3 independent experiments normalized to GAPDH. Error bars represent the standard deviation. e) Western blot showing the reduced level of E-cadherin in siRNA generated E-cadherin KD cell line for MCF7A cells. f and g) Average flow field for MCF7A control cells (n = 2047 defects from 3 independent experiments) (f) and siRNA E-cadherin KD MCF7A cells (n = 1256 defects from 3 independent experiments) (f). (h) Uncropped blots of all the western blots shown so far.

Article Snippet: Primary antibodies were diluted in PBS Tween at E-cadherin (24E10-1:1000 for MDCK cells) (Santa Cruz, SC7870-1:200 for MCF7 cells), α-catenin (1:1000), β -catenin (1:1000), vinculin (gift from Marina Glukhova, 1:500), GAPDH (Protein Tech Europe 60004-1 for MDCK cells and abcam- 1:5000, ab181603 for MCF7 cells- 1:500), alpha-tubulin (1:5000) (Sigma T9026) overnight on a shaker at 4 °C.

Techniques: Immunofluorescence, Staining, Western Blot, Standard Deviation, Generated, Control

Journal: Nature materials

Article Title: Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers

doi: 10.1038/s41563-021-00919-2

Figure Lengend Snippet: a) Average yy - and xy -components of strain rate map around comet ( +1/2 ) defect obtained from experiments (left and middle respectively) and corresponding average flow field (right) ( n = 1767 defects from 2 independent experiments) for MDCK E-cadherin KO cells rescued with E-cadherin GFP. b) Total number of defects obtained per 0.55mm2 as a function of time on MDCK WT and MDCK E-cadherin KO monolayers. (n=10) from 2 independent experiments. c) Average vorticity and velocity field around trefoil (-1/2) defects in WT (left) (n=1934) and E-cadherin KO (right) (n=2028) monolayers. d) Average vorticity and velocity field around trefoil (-1/2) defects in control (left) (n=3200) and condition without active intercellular forces (right)(n=3200) monolayers obtained from simulations. e) Mean square displacement (MSD) plotted against time lag for comet (+1/2) and trefoil (-1/2) defects obtained from MDCK WT and MDCK E-cadherin KO monolayers (n=11). Error bars represent the standard deviation.

Article Snippet: Primary antibodies were diluted in PBS Tween at E-cadherin (24E10-1:1000 for MDCK cells) (Santa Cruz, SC7870-1:200 for MCF7 cells), α-catenin (1:1000), β -catenin (1:1000), vinculin (gift from Marina Glukhova, 1:500), GAPDH (Protein Tech Europe 60004-1 for MDCK cells and abcam- 1:5000, ab181603 for MCF7 cells- 1:500), alpha-tubulin (1:5000) (Sigma T9026) overnight on a shaker at 4 °C.

Techniques: Control, Standard Deviation

Journal: Nature materials

Article Title: Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers

doi: 10.1038/s41563-021-00919-2

Figure Lengend Snippet: a) Mean traction force for both MDCK WT (n=31) and MDCK Ecadherin KO cells (n=27). b) Average yy- and xy-components of strain rate map around comet (+1/2) defect obtained from experiments (left and middle respectively) and corresponding average velocity flow field (right) (n = 1428 defects from 2 independent experiments) for MDCK E-cadherin KO cells plated on PDMS substrates of stiffness 15kPa from which stress maps were obtained in ’.

Article Snippet: Primary antibodies were diluted in PBS Tween at E-cadherin (24E10-1:1000 for MDCK cells) (Santa Cruz, SC7870-1:200 for MCF7 cells), α-catenin (1:1000), β -catenin (1:1000), vinculin (gift from Marina Glukhova, 1:500), GAPDH (Protein Tech Europe 60004-1 for MDCK cells and abcam- 1:5000, ab181603 for MCF7 cells- 1:500), alpha-tubulin (1:5000) (Sigma T9026) overnight on a shaker at 4 °C.

Techniques:

Journal: Nature materials

Article Title: Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers

doi: 10.1038/s41563-021-00919-2

Figure Lengend Snippet: a) pMRLC (left), zoom of pMRLC (middle), actin (right) staining of MDCK WT (top) and E-cadherin KO (bottom) monolayers. b) Evolution of mean traction force of MDCK WT and E-cadherin KO monolayers before and after 20μM blebbistatin treatment (n=10 from 2 independent experiments). c, d, e) actin (red) and paxillin (green) (c), vinculin (d), YAP (green), and nucleus (blue) (e), staining within a monolayer for both MDCK WT and E-cadherin KO cells. c) Area of focal adhesion (left) and length of focal adhesion within the monolayer for n = 106 focal adhesions. d) Mean intensity of vinculin at the cell-cell junction in the middle plane ( n = 54 ). e) Distribution of YAP in nucleus, cytoplasm, or uniform distribution calculated for n = 1162 cells (MDCK WT) and n = 1008 cells (MDCK E-cadherin KO). Error bars represent the standard deviation. Unpaired t-test was performed leading to *p<0.05 , **p<0.01 , ***p<0.001 and ****p<0.0001 . Scale bars, 20μm and 10μm for the figure zooms.

Article Snippet: Primary antibodies were diluted in PBS Tween at E-cadherin (24E10-1:1000 for MDCK cells) (Santa Cruz, SC7870-1:200 for MCF7 cells), α-catenin (1:1000), β -catenin (1:1000), vinculin (gift from Marina Glukhova, 1:500), GAPDH (Protein Tech Europe 60004-1 for MDCK cells and abcam- 1:5000, ab181603 for MCF7 cells- 1:500), alpha-tubulin (1:5000) (Sigma T9026) overnight on a shaker at 4 °C.

Techniques: Staining, Standard Deviation

Journal: Nature materials

Article Title: Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers

doi: 10.1038/s41563-021-00919-2

Figure Lengend Snippet: a, b, c) Average yy - and xy -components of strain rate map around +1/2 defect obtained from experiments (left and middle respectively) and corresponding average velocity flow field (right) for MDCK E-cadherin KO cells treated with 5μM blebbistatin (a) ( n = 2174 defects from 2 independent experiments), 20μM blebbistatin (b) ( n = 1223 defects from 2 independent experiments), and 25μM Y27632 (c) (n = 1965 defects from 2 independent experiments). d, e) Average yy- and xy components of strain rate map around +1/2 defect obtained from experiments (left and middle respectively) and corresponding average velocity flow field (right) for MDCK WT cells treated with 20μM blebbistatin (d) ( n = 1287 defects from 2 independent experiments), and 25μM Y27632 (e) ( n = 2472 defects from 2 independent experiments).

Article Snippet: Primary antibodies were diluted in PBS Tween at E-cadherin (24E10-1:1000 for MDCK cells) (Santa Cruz, SC7870-1:200 for MCF7 cells), α-catenin (1:1000), β -catenin (1:1000), vinculin (gift from Marina Glukhova, 1:500), GAPDH (Protein Tech Europe 60004-1 for MDCK cells and abcam- 1:5000, ab181603 for MCF7 cells- 1:500), alpha-tubulin (1:5000) (Sigma T9026) overnight on a shaker at 4 °C.

Techniques:

Journal: Nature materials

Article Title: Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers

doi: 10.1038/s41563-021-00919-2

Figure Lengend Snippet: a) Immunostaining of basal plane of vinculin (left), paxillin (middle) and merge (right) in MDCK WT (top) and MDCK E-cadherin KO monolayers. b) Intensity of vinculin plotted against paxillin for n=15 focal adhesions in MDCK WT and n=16 focal adhesions in MDCK Ecadherin KO monolayers. Scale bars: 20μm.

Article Snippet: Primary antibodies were diluted in PBS Tween at E-cadherin (24E10-1:1000 for MDCK cells) (Santa Cruz, SC7870-1:200 for MCF7 cells), α-catenin (1:1000), β -catenin (1:1000), vinculin (gift from Marina Glukhova, 1:500), GAPDH (Protein Tech Europe 60004-1 for MDCK cells and abcam- 1:5000, ab181603 for MCF7 cells- 1:500), alpha-tubulin (1:5000) (Sigma T9026) overnight on a shaker at 4 °C.

Techniques: Immunostaining

Journal: Nature materials

Article Title: Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers

doi: 10.1038/s41563-021-00919-2

Figure Lengend Snippet: a, b) Average yy- and xy-components of strain rate map around +1/2 defect obtained from experiments (left and middle respectively) and corresponding average flow field (right) for MDCK WT cells (a) (n = 1426 defects from 2 independent experiments) and E-cadherin KO cells (b) (n = 1041 defects from 2 independent experiments).

Article Snippet: Primary antibodies were diluted in PBS Tween at E-cadherin (24E10-1:1000 for MDCK cells) (Santa Cruz, SC7870-1:200 for MCF7 cells), α-catenin (1:1000), β -catenin (1:1000), vinculin (gift from Marina Glukhova, 1:500), GAPDH (Protein Tech Europe 60004-1 for MDCK cells and abcam- 1:5000, ab181603 for MCF7 cells- 1:500), alpha-tubulin (1:5000) (Sigma T9026) overnight on a shaker at 4 °C.

Techniques:

Journal: Nature materials

Article Title: Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers

doi: 10.1038/s41563-021-00919-2

Figure Lengend Snippet: a) Phase diagram showing the transition of extensile and contractile behaviour with varying values of intercellular and intracellular stresses obtained from simulations. b) Phase separation (demixing) observed from simulations where the contractile particles (orange) are surrounded by extensile particles (green). c) Cell sorting (demixing) observed for a mixture of MDCK WT and MDCK E-cadherin KO cells, where WT cells are surrounded by E-cadherin KO cells (E-cadherin, green, cadherin 6, red, actin, black). XZ and YZ projection show the height difference between the two cells when mixed. Scale bars, 20μm . d,e) Early stages of cell sorting when MDCK WT (magenta) and MDCK E-cadherin KO (green) monolayers are mixed at 30-70 (d) and 70-30 (e) ratio. Scale bars: 100μm .

Article Snippet: Primary antibodies were diluted in PBS Tween at E-cadherin (24E10-1:1000 for MDCK cells) (Santa Cruz, SC7870-1:200 for MCF7 cells), α-catenin (1:1000), β -catenin (1:1000), vinculin (gift from Marina Glukhova, 1:500), GAPDH (Protein Tech Europe 60004-1 for MDCK cells and abcam- 1:5000, ab181603 for MCF7 cells- 1:500), alpha-tubulin (1:5000) (Sigma T9026) overnight on a shaker at 4 °C.

Techniques: FACS

Journal: Nature materials

Article Title: Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers

doi: 10.1038/s41563-021-00919-2

Figure Lengend Snippet: a,b) Time lapse sorting of extensile and contractile cells observed over time represented by mixing index in simulations (a) and experiments (b) of MDCK WT (magenta) and E-cadherin KO cells tagged with LifeAct GFP (green). In (a) ζs/Rα = 0.042, ζQ/Rα = -0.062 for the extensile cells and ζs/Rα = 0.0, ζQ/Rα = -0.062 for the contractile cells. Mixing index was obtained from two independent simulations and the error bars mark the standard deviation. Mixing index in experiments (b) was obtained from n=5 different clusters from 2 independent samples. Error bars represent the standard deviation. Scale bars: 100μm .

Article Snippet: Primary antibodies were diluted in PBS Tween at E-cadherin (24E10-1:1000 for MDCK cells) (Santa Cruz, SC7870-1:200 for MCF7 cells), α-catenin (1:1000), β -catenin (1:1000), vinculin (gift from Marina Glukhova, 1:500), GAPDH (Protein Tech Europe 60004-1 for MDCK cells and abcam- 1:5000, ab181603 for MCF7 cells- 1:500), alpha-tubulin (1:5000) (Sigma T9026) overnight on a shaker at 4 °C.

Techniques: Standard Deviation

Journal: Nature materials

Article Title: Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers

doi: 10.1038/s41563-021-00919-2

Figure Lengend Snippet: a) Demixing of MDCK WT and E-cadherin KO at different starting densities, WT (30%) and E-cadherin KO (70%) (left) and WT (70%) and E-cadherin KO (30%) (right). b) Demixing of extensile and contractile particles obtained from simulations at different starting densities. Extensile and contractile particles are mixed at 50-50 (left), 30-70 (middle) and 70-30 (right) respectively. In (b) ζs/Rα = 0.016, ζQ/Rα = -0.016 for the extensile cells and ζs/Rα = 0.0, ζQ/Rα = -0.016 for the contractile cells. c) Demixing phase observed before and after the addition of 20μM blebbistatin characterized by mixing index (left) (n=5) and circularity of several colonies (right) (n=5). Error bars represent the standard deviation. Scale bars: 100μm .

Article Snippet: Primary antibodies were diluted in PBS Tween at E-cadherin (24E10-1:1000 for MDCK cells) (Santa Cruz, SC7870-1:200 for MCF7 cells), α-catenin (1:1000), β -catenin (1:1000), vinculin (gift from Marina Glukhova, 1:500), GAPDH (Protein Tech Europe 60004-1 for MDCK cells and abcam- 1:5000, ab181603 for MCF7 cells- 1:500), alpha-tubulin (1:5000) (Sigma T9026) overnight on a shaker at 4 °C.

Techniques: Standard Deviation

Journal: Molecular pharmaceutics

Article Title: Targeting Triple Negative Breast Cancer with a Nucleus-Directed p53 Tetramerization Domain Peptide

doi: 10.1021/acs.molpharmaceut.0c00978

Figure Lengend Snippet: Cy5p53Tet penetrates into MDA-MB-468 TNBC cells expressing mtp53 R273H. (A) Schematic of the structure of Cy5p53Tet. (B) Live cell imaging staining of MCF7 and MDA-MB-468 cells after 2 h of incubation with 500 nM Cy5p53Tet (red). Hoechst staining (blue) was used to stain the nuclei. Two independent experiments with biological replicates were performed. (C) p53 protein levels in MCF7 and MDA-MB-468 cells determined by Western blot analysis before carrying out live cell imaging. (D) Quantification of Cy5p53Tet uptake in MCF7 and MDA-MB-468 cells via Nikon Element analysis. At least 200 cells per sample were measured by fluorescence microscopy. (E) Flow cytometry of MCF7 and MDA-MB-468 cells after incubation with 100 or 500 nM Cy5p53Tet for 2 h at 37 °C. FlowJo software was used to analyze the cytometric data. (F) Geometric MFI from the FACS experiments in (E). (G) MTT assay conducted in MCF7, MDA-MB-468, HCC70, SK-BR-3, and MCF10A cells to measure mitochondrial dehydrogenase activity in response to 500 nM Cy5p53Tet treatment for 24 h. Three independent experiments with biological replicates were performed for all ± SEM *p-value ≤ 0.05, **p-value ≤ 0.01, ***p-value ≤ 0.001.

Article Snippet: Human breast cancer cell lines MCF7, MDA-MB-468, MDA-MB-231, HCC70, and SK-BR-3 and normal human mammary epithelial cell MCF10A were purchased from American Type Culture Collection (ATCC).

Techniques: Expressing, Live Cell Imaging, Staining, Incubation, Western Blot, Fluorescence, Microscopy, Flow Cytometry, Software, MTT Assay, Activity Assay