Journal: Nature Communications

Article Title: Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity

doi: 10.1038/ncomms13998

Figure Lengend Snippet: ( a , b ) Distribution of myosin-IIa and -IIb in control and EpCAM-depleted cells. Scale bars, 5 μm. ( c , d ) Distribution of E-cadherin and myosin-IIa, or E-cadherin and myosin-IIb in EpCAM-depleted cells. Scale bars, 5 μm. ( e , f ) 3D-SIM analysis of E-cadherin and myosin-IIb, or Crb3 and myosin-IIa in EpCAM-deprived cells. Scale bars, 5 μm. ( g ) Western blot analysis of MLC2 and P-MLC2 amounts in control and EpCAM-depleted cells. GAPDH was used as loading control. ( h ) Quantification of P-MLC2 amount relative to MLC2 in control and EpCAM-depleted cells. One-way ANOVA with Dunnett's test, * P <0.01. Caco2shNT=1, Caco2shEpCAM#1=0.11±0.2, Caco2shEpCAM#2=0.42±0.2. ( i ) Distribution of P-MLC2 in control or EpCAM-silenced cells. Scale bars, 5 μm. ( j ) Quantification of total P-MLC2 immunofluorescence intensity relative to MLC2 immunofluorescence intensity in control or EpCAM-depleted cells. One-way analysis of variance test with Dunnett's test, * P =0.007, ** P =0.002. n (Caco2shNT) =30 cells, n (Caco2shEpCAM#1) =30, n (Caco2shEpCAM#2) =30. Caco2shNT=1, Caco2shEpCAM#1=0.4±0.05, Caco2shEpCAM#2=0.52±0.07. ( k ) Quantification of P-MLC2 immunofluorescence intensity at TCs relative to immunofluorescence intensity at bicellular junctions (BJs) in control or EpCAM-depleted cells. One-way analysis of variance with unpaired t -test, * P =0.0001. n (Caco2shNT) =30 cells, n (Caco2shEpCAM#1) =28, n (Caco2shEpCAM#2) =30. Caco2shNT=1, Caco2shEpCAM#1=6.01±1.24, Caco2shEpCAM#2=4.1±0.33. ( l – n ) Distribution of myosin-IIa, -IIb and P-MLC2 in control or CTE biopsies. Scale bars, 5 μm. N (Control) =3 biopsies, N (CTE) =3. ( o – q ) Quantification of myosin-IIa, -IIb or P-MLC2 immunofluorescence intensity at TCs relative to immunofluorescence intensity at BJs in control or CTE biopsies. N (Control) =3 biopsies, N (CTE) =3. Unpaired t -test, ( o ) * P <0.0001, ( p ) * P =0.0062, ( q ) * P =0.0016. n (Control myosin-IIa) =37 cells, n (CTE myosin-IIa) =29, n (Control myosin-IIb) =14, n (CTE myosin-IIb) =14, n (Control P-MLC2) =15, n (CTE P-MLC2) =21. Myosin-IIa (Control) =1±0.04, myosin-IIa (CTE) =5.86±0.54, myosin-IIb (Control) =1±0.08, myosin-IIb (CTE) =2.27±0.39, P-MLC2 (Control) =1±0.07, P-MLC2 (CTE) =3.25±0.62. For quantification, three independent replicates have been performed. Values are mean±s.e.m. Nuclei were stained with Hoechst 33342.

Article Snippet: Rabbit polyclonal antibody directed against P-MLC2 (S19/S20, #600-401-416, IF dilution 1:100, WB dilution 1:500) was from Rockland (Limerick, PA, USA).

Techniques: Control, Western Blot, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity

doi: 10.1038/ncomms13998

Figure Lengend Snippet: ( a ) Quantification of P-MLC2 immunofluorescence intensity at TCs relative to immunofluorescence intensity at BJs in control, EpCAM-depleted or 2h-blebbistatin-treated EpCAM-depleted cells. One-way analysis of variance with unpaired t -test, * P <0.05. n (Caco2shNT) =30 cells, n (Caco2shEpCAM#1) =28, n (Caco2shEpCAM#2) =30, n (Caco2shEpCAM#1+Blebbistatin 50 μM) =31, n (Caco2shEpCAM#2+Blebbistatin 50 μM) =30. Caco2shNT=1, Caco2shEpCAM#1=6.01±1.24, Caco2shEpCAM#2=4.1±0.33, Caco2shEpCAM#1+Blebbistatin 50 μM=1.57±0.17, Caco2shEpCAM#2+Blebbistatin50 μM=0.91±0.06. ( b ) SEM analyses of apical surfaces of DMSO-treated control, EpCAM-depleted or EpCAM-depleted cells treated with blebbistatin for 2 h. Scale bars, 4 μm. ( c ) Confocal analysis and 3D rendering of z -stacks of actin location at TCs in DMSO-treated control, EpCAM-depleted or EpCAM-depleted cells treated with blebbistatin for 2 h. Scale bars, 4 μm. ( d ) High magnifications of EADs from SEM or confocal and 3D rendering analyses. Scale bars, 1 μm. ( e ) Quantification of EADs on DMSO or blebbistatin treatment in Caco2shNT or shEpCAM cells. One-way ANOVA with Tukey's test, **** P <0.0001. n (Caco2shNT+DMSO) =107 cells, n (Cao2shEpCAM#1+DMSO) =105, n (Caco2shEpCAM#2+DMSO) =91, n (Caco2shEpCAM#1+Blebbistatin) =96, n (Caco2shEpCAM#2+Blebbistatin) =81. EAD-positive Caco2shNT cells=0%, EAD-positive Caco2shEpCAM#1+DMSO cells=87.41±1.64%, EAD-positive Caco2shEpCAM#1+DMSO cells=91.98±4.21%, EAD-positive Caco2shEpCAM#1+blebbistatin cells=9.55±5.46%, EAD-positive Caco2shEpCAM#1+DMSO=18.43±1.83%. ( f ) Confocal analysis of actin distribution and brush border formation in Caco2shEpCAM cells. Cells were treated for 2 h with DMSO, or for 10 min or 2 h with 5 or 50 μM of blebbistatin. Scale bars, 5 μm. ( g ) Quantification of EADs on DMSO or blebbistatin treatment in Caco2 shEpCAM cells. N (2 h, DMSO)=5,467 cells, N (10 min, 5 μΜ)=3,598, N (10 min, 50 μΜ)=4,032, N (2 h, 5 μΜ)=289, N (2 h, 50 μΜ)=4,416. t -test, P <0.0001. ( h ) Analysis of lateral membrane behaviour in EpCAM-deprived Caco2 cells during 50 μM blebbistatin treatment and time-lapse acquisitions. Lateral membranes have been stained with SirActin labelling. Scale bars, 10 μm. For quantification, three independent replicates have been performed. Values are mean±s.e.m. Nuclei were stained with Hoechst 33342.

Article Snippet: Rabbit polyclonal antibody directed against P-MLC2 (S19/S20, #600-401-416, IF dilution 1:100, WB dilution 1:500) was from Rockland (Limerick, PA, USA).

Techniques: Immunofluorescence, Control, Membrane, Staining

Journal: Nature Communications

Article Title: Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity

doi: 10.1038/ncomms13998

Figure Lengend Snippet: ( a ) Statistical analysis of the number of tuft-like structures detected along the 3D microfabricated villus structures in control ( Caco2 shNT ), EpCAM-depleted cells ( Caco2 shEpCAM ) and 2h- blebbistatin-treated EpCAM-depleted cells ( Caco2 shEpCAM#1+Blebbistatin 50 μ M ). Three independent replicates have been performed. One-way analysis of variance with unpaired t -test, * P <0.0001. n (Caco2 shNT) =196 villi, n (Caco2 shEpCAM) =210 villi, n (Caco2 shEpCAM+Blebistatin) =210 villi. Caco2 shNT(mean number of tufts per villus)=5, Caco2 shEpCAM(mean number of tufts per villus)=12, Caco2 shEpCAM+Blebbistatin 50 μM(mean number of tufts per villus)=6. ( b – d ) Confocal microscopy analysis of myosin-IIa ( b ), myosin-IIb ( c ) and P-MLC2 ( d ) distribution in control ( Caco2 shNT ) and EpCAM-silenced ( Caco2 shEpCAM ) cells that were grown on villous PDMS inserts or in 3D Matrigel cultures for 21 days. Transversal xy views are presented. Scale bars, 50 μm. ( e ) Schemes recapitulating the cellular and epithelial phenotypes observed in control ( Caco2 shNT ) or EpCAM-depleted ( Caco2 shEpCAM ) conditions when cells are grown in 2D, 3D synthetic villi or 3D Matrigel cultures. Contractile apparatus (blue) and predictated tensile forces (red arrows) are presented.

Article Snippet: Rabbit polyclonal antibody directed against P-MLC2 (S19/S20, #600-401-416, IF dilution 1:100, WB dilution 1:500) was from Rockland (Limerick, PA, USA).

Techniques: Control, Confocal Microscopy