Journal: eLife

Article Title: Rho-ROCK liberates sequestered claudin for rapid de novo tight junction formation

doi: 10.7554/eLife.102794

Figure Lengend Snippet: ( A ) Schematic showing the elimination of an apoptotic cell by apical extrusion and de novo tight junction (TJ) formation. Tight junctions are established between newly adjacent cells (shown in green) concurrent with extrusion of the apoptotic cell. The TJs between the apoptotic cell and the cells that affect extrusion (shown in purple) gradually diminish as the new TJs mature, ensuring that the epithelial barrier is never disrupted. ( B ) Live cell images of EpH4 cells expressing GFP-claudin-3 in cells neighboring laser-wounded cell. Cell marked by an asterisk was wounded at time zero. The schematics represent the cells before laser irradiation and after apical extrusion. Purple arrowheads indicate the old TJ with the apoptotic cell and green arrowheads indicate the newly assembled TJ. The upper images (time = 60 and 80 min) show higher magnification corresponding to each time point. The right panels are orthogonal views. Scale bar = 20 μm. (See also ). ( C ) Immunofluorescence images showing apical extrusion at 60 min post-laser irradiation. Cells were stained with anti-claudin-3 pAb (green) and either anti-ZO-1 mAb (magenta, upper) or anti-occludin mAb (magenta, lower). Purple arrowheads indicate old TJs with the apoptotic cell and green arrowheads indicate newly assembled TJs. The bottom panels represent orthogonal views. Scale bar = 10 μm. ( D ) Live cell images of cells surrounding a laser-wounded cell expressing GFP-AnillinC as a probe for active RhoA. Cell marked by an asterisk was wounded at 0 min. The arrowheads indicate accumulation of active RhoA at the junction between an apoptotic cell and a neighboring cell (white) or between newly adjacent cells (yellow). Scale bar = 20 μm. (See also ). ( E ) Immunofluorescence images showing apical extrusion at 60 min post-laser irradiation. Cells were stained with phosphorylated myosin light chain (ppMLC) mAb (magenta) and anti-NMIIB mAb (green). The asterisk in the X-Z image indicates the apoptotic cell. Scale bar = 10 μm. ( F ) Live cell images of cells surrounding a laser-wounded cell co-expressing GFP-claudin-3 and mScarlet-PLCδPH (membrane marker). Cells were pre-treated with H1152 and the cell marked by an asterisk was wounded at 0 min. The filled purple arrowhead indicates a TJ with an apoptotic cell and the unfilled purple arrowheads indicate the gradual dissolution of said TJ. Filled purple arrowheads indicate TJ between an apoptotic cell and neighboring cells, while unfilled purple arrowheads indicate the disappearance of TJ. Images of scarlet-PLCδ PH are projections of basal confocal slices and the extending lamellipodia are indicated by the white arrowheads. The inset enlarged at right—and illustrated below—shows the absence of new TJs among the now adjacent cells A-E. Scale bar = 20 µm. (See also ). ( G ) Bar graph showing the paracellular flux of 70 kDa FITC-dextran tracer molecule at 9 hr post-doxorubicin treatment (4 µM) (control: N=3, H1152: N=5; error bar: ± SD; Mann–Whitney U test). ( H ) Schematic illustrating the formation of new TJs between neighbor cells concurrent with extrusion of the apoptotic cell. The formation of these TJ requires the activation of RhoA.

Article Snippet: The following primary antibodies were used for immunofluorescence microscopy and immunoblotting: rabbit anti-claudin-3 pAb (34–1700; Thermo Fisher Scientific); mouse anti-claudin-4 mAb (32–9400; Thermo Fisher Scientific); and rabbit anti-claudin-7 pAb (34–9100; Thermo Fisher Scientific); rabbit anti-α-catenin pAb (C2081; Sigma Aldrich); rabbit anti-claudin-1 pAb (SAB4200534; Sigma Aldrich); rat anti-E-cadherin mAb (ECCD2; Takara Bio); rabbit anti-RhoA mAb (2117; Cell Signaling Technology); rabbit anti-phospho-Myosin Light Chain 2 (Thr18/Thr19) mAb (95777; Cell Signaling Technology); rabbit anti-TROP2 mAb (ab214488; abcam); rabbit anti-EpCAM pAb (ab71916; abcam); mouse anti-GM130 mAb (610822; BD).

Techniques: Expressing, Irradiation, Immunofluorescence, Staining, Membrane, Marker, Dissolution, Control, MANN-WHITNEY, Activation Assay

Journal: eLife

Article Title: Rho-ROCK liberates sequestered claudin for rapid de novo tight junction formation

doi: 10.7554/eLife.102794

Figure Lengend Snippet: ( A and B ) RhoA activities in undifferentiated and differentiated HaCaT cells. Cells were lysed and subjected to a GST-Rhotekin pulldown assay. Total cell lysates and precipitates were analyzed by immunoblotting with anti-RhoA mAb. Molecular weight measurements are in kDa. ( B ) A graph illustrating the rate of RhoA activities. RhoA activity was calculated by dividing the intensity of activated RhoA (pulldown signal) by the intensity of input signal (N=6; error bar: ± SD; Student’s t-test). ( C and D ) HaCaT cells were cultured in normal medium or Ca +JNK inh medium for 24 hr, fixed, and then stained with anti-ppMLC mAb (green) and anti–E-cadherin mAb (magenta). Scale bar: 20 μm. Graph showing the quantification of junctional enrichment of phosphorylated myosin light chain (ppMLC) signals ( D ). The quantification methods are illustrated in and details are described in Methods (control: N=5, Ca +JNK inh: N=8; error bar: ± SD; Student’s t test). ( E ) Mouse ear whole-mount immunofluorescence analysis for ppMLC, ZO-1, and E-cadherin. The asterisk indicates the nucleus of cells belonging to the indicated layer. ( F and G ) HaCaT cells expressing GFP-claudin-3 were cultured in normal medium or Ca +JNK inh medium supplemented with DMSO (control) or Y27632 (ROCK inhibitor) for 24 hr, fixed, and then stained with anti–E-cadherin mAb (magenta). Yellow arrowheads indicate TJs. Scale bar: 20 μm. ( G ) A bar graph illustrating the junctional enrichment of claudin-3 after differentiation (control: N=5, Ca +JNK inh: N=6; error bar:± SD; Student’s t-test). ( H ) Schematic illustrating TJ formation in the epidermal granular layer. In the epidermis, all keratinocytes of basal, spinous, and granular layers express claudin-1. However, functional TJs are only formed in the SG2 layer. The activation of the Rho-ROCK pathway is crucial for the formation of TJs in the SG2 layer. Figure 3—source data 1. Full blot original data for the western blots shown in . Figure 3—source data 2. Full blot data for the western blots shown in .

Article Snippet: The following primary antibodies were used for immunofluorescence microscopy and immunoblotting: rabbit anti-claudin-3 pAb (34–1700; Thermo Fisher Scientific); mouse anti-claudin-4 mAb (32–9400; Thermo Fisher Scientific); and rabbit anti-claudin-7 pAb (34–9100; Thermo Fisher Scientific); rabbit anti-α-catenin pAb (C2081; Sigma Aldrich); rabbit anti-claudin-1 pAb (SAB4200534; Sigma Aldrich); rat anti-E-cadherin mAb (ECCD2; Takara Bio); rabbit anti-RhoA mAb (2117; Cell Signaling Technology); rabbit anti-phospho-Myosin Light Chain 2 (Thr18/Thr19) mAb (95777; Cell Signaling Technology); rabbit anti-TROP2 mAb (ab214488; abcam); rabbit anti-EpCAM pAb (ab71916; abcam); mouse anti-GM130 mAb (610822; BD).

Techniques: Western Blot, Molecular Weight, Activity Assay, Cell Culture, Staining, Control, Immunofluorescence, Expressing, Functional Assay, Activation Assay

Journal: eLife

Article Title: Rho-ROCK liberates sequestered claudin for rapid de novo tight junction formation

doi: 10.7554/eLife.102794

Figure Lengend Snippet: ( A ) Undifferentiated HaCaT cells expressing constitutive active RhoA (RhoA CA) were stained with anti-claudin-3 pAb (green), and phalloidin (grayscale). The arrowheads indicate ectopic tight junctions. Scale bar: 20 μm. ( B ) Undifferentiated HaCaT cells expressing RhoA CA were stained with anti-claudin-3 pAb (magenta) and either anti-ZO-1 mAb (green; upper) or anti-occludin mAb (green; lower). The arrowheads indicate ectopic TJs. Scale bar: 20 μm. ( C and D ) Confluent undifferentiated HaCaT cells were cultured in serum-free medium for 24 hr, treated with a recombinant bacterial cytotoxic necrotizing factor (CNF) toxin (CN-03), also known as Rho activator II (1 μg/ml) for 2 h, fixed, and then stained with anti-claudin-3 pAb (green), anti-E-cadherin mAb (magenta), and phalloidin (grayscale). Scale bar: 20 μm. ( D ) An alternate field of view corresponding to the experiment in ( C ). ( E and F ) Confluent undifferentiated HaCaT cells were treated with DMSO (control) or Narciclasine (100 nM; ROCK activator) for 4 hr, fixed, and then stained with anti-claudin-3 pAb (green), anti-E-cadherin mAb (magenta), and phalloidin (grayscale). Scale bar: 20 μm. ( F ) An alternate field of view of the experiment in ( E ). ( G ) Junctional enrichment of claudin-3 was quantified based on the method described in (N=6; error bar: ± SD; Tukey-Kramer One-way Anova). ( H and I ) Western blot of Triton X-100–soluble fractions and insoluble fractions from HaCaT cells treated with Rho activator (CN-03) or Narciclasine. The proportion of insoluble claudin-1 was quantified in ( I ) (N=5; error bar: ± SD; Kruskal-Wallis test followed by Steel-Dwass post hoc test; *p<0.05). Figure 4—source data 1. Full blot original image for the western blots shown in . Figure 4—source data 2. Full blot data for the western blots shown in .

Article Snippet: The following primary antibodies were used for immunofluorescence microscopy and immunoblotting: rabbit anti-claudin-3 pAb (34–1700; Thermo Fisher Scientific); mouse anti-claudin-4 mAb (32–9400; Thermo Fisher Scientific); and rabbit anti-claudin-7 pAb (34–9100; Thermo Fisher Scientific); rabbit anti-α-catenin pAb (C2081; Sigma Aldrich); rabbit anti-claudin-1 pAb (SAB4200534; Sigma Aldrich); rat anti-E-cadherin mAb (ECCD2; Takara Bio); rabbit anti-RhoA mAb (2117; Cell Signaling Technology); rabbit anti-phospho-Myosin Light Chain 2 (Thr18/Thr19) mAb (95777; Cell Signaling Technology); rabbit anti-TROP2 mAb (ab214488; abcam); rabbit anti-EpCAM pAb (ab71916; abcam); mouse anti-GM130 mAb (610822; BD).

Techniques: Expressing, Staining, Cell Culture, Recombinant, Control, Western Blot

Journal: eLife

Article Title: Rho-ROCK liberates sequestered claudin for rapid de novo tight junction formation

doi: 10.7554/eLife.102794

Figure Lengend Snippet: ( A and B ) Whole-cell lysates of wild-type (WT) treated with Rho activator (CN-03), Narciclasine, or acid medium (pH 6.0) were immunoblotted with the indicated antibodies. Molecular weight measurements are in kDa. Schematic showing the sequential activation of matriptase. M24 mAb detects total Matriptase and M69 mAb the activated form. ( B ) Quantification of the cleaved TROP2 fragment relative to total TROP2. (N=4; error bar: ± SD; Kruskal-Wallis test followed by Steel-Dwass post hoc test; *p<0.05). ( C and D ) Confluent undifferentiated HaCaT cells were cultured in normal (control) or acid medium (pH 6.0) for 20 min, fixed, and then stained with anti-claudin-3 pAb (green), anti-E-cadherin mAb (magenta), and phalloidin (grayscale). Scale bar = 20 μm. ( D ) A bar graph illustrating the junctional enrichment of claudin-3 (N=6; error bar: ± SD; Student’s t-test). ( E and F ) Western blot of Triton X-100–soluble and insoluble fractions from confluent undifferentiated HaCaT cells cultured in acid medium. The proportion of insoluble claudin-1 was quantified in ( F ) (N=5; error bar: ± SD; Student’s t-test). ( G ) Undifferentiated HaCaT cells treated with DMSO (control) or Camostat (serine protease inhibitor) expressing constitutive active RhoA (Rho CA) were fixed and stained with anti-claudin-3 pAb (green) and phalloidin (grayscale). Scale bar: 20 μm. ( H ) Bar graph illustrating the extent to which the matriptase inhibitor Camostat negated the Narciclasine-induced claudin enrichment at cell-cell contacts. After inhibition of serine protease with Camostat, confluent undifferentiated HaCaT cells were treated with Narciclasine (100 nM; ROCK activator) for 4 hr and stained to quantify the junctional enrichment of claudin-3 (N=6; error bar: ± SD; Student’s t-test). ( I ) Undifferentiated HaCaT cells expressing constitutive active RhoA (Rho CA) were stained with anti-TROP2 mAb (green) and claudin-4 mAb (magenta). Insets are enlarged images. Scale bar: 20 μm. Figure 5—source data 1. Full blot original images for the western blots shown in . Figure 5—source data 2. Full blot data for the western blots shown in .

Article Snippet: The following primary antibodies were used for immunofluorescence microscopy and immunoblotting: rabbit anti-claudin-3 pAb (34–1700; Thermo Fisher Scientific); mouse anti-claudin-4 mAb (32–9400; Thermo Fisher Scientific); and rabbit anti-claudin-7 pAb (34–9100; Thermo Fisher Scientific); rabbit anti-α-catenin pAb (C2081; Sigma Aldrich); rabbit anti-claudin-1 pAb (SAB4200534; Sigma Aldrich); rat anti-E-cadherin mAb (ECCD2; Takara Bio); rabbit anti-RhoA mAb (2117; Cell Signaling Technology); rabbit anti-phospho-Myosin Light Chain 2 (Thr18/Thr19) mAb (95777; Cell Signaling Technology); rabbit anti-TROP2 mAb (ab214488; abcam); rabbit anti-EpCAM pAb (ab71916; abcam); mouse anti-GM130 mAb (610822; BD).

Techniques: Molecular Weight, Activation Assay, Cell Culture, Control, Staining, Western Blot, Protease Inhibitor, Expressing, Inhibition

Journal: eLife

Article Title: Rho-ROCK liberates sequestered claudin for rapid de novo tight junction formation

doi: 10.7554/eLife.102794

Figure Lengend Snippet: ( A and B ) Whole-cell lysates of wild-type (WT) and TROP2 KO HaCaT cells were immunoblotted with the indicated antibodies. Molecular weight measurements are in kDa. The expression level of claudin-1 normalized to α-tubulin was quantified in ( B ) (N=4; error bar: ± SD; Student’s t test). ( C and D ) Representative western blots of surface and total amount of claudin-1. Molecular weight measurements are in kDa. The graph of ( D ) illustrates the quantification of claudin-1 present on the cell surface relative to the total amount (N=3; error bar: ± SD; Student’s t test). ( E ) Undifferentiated TROP2 KO HaCaT cells were stained with anti-claudin-1 pAb (green) and anti-GM130 mAb (magenta; Golgi marker). Insets are enlarged images. Scale bar: 20 μm. ( F ) Undifferentiated WT or TROP2 KO HaCaT cells expressing constitutive active RhoA (Rho CA) were stained with anti-claudin-3 pAb (green) and phalloidin (grayscale). Scale bar: 20 μm. ( G ) Undifferentiated WT and TROP2 KO HaCaT cells were co-cultured, incubated in acid buffer (pH 6.0) for 20 min, fixed, and then stained with anti-claudin-4 mAb (green) and anti-TROP2 mAb (magenta). Dotted line overlays the border between WT and TROP2 KO cells. Scale bar: 20 μm. ( H ) Bar graph illustrating the impairment of either Narciclasine- or acid medium-induced claudin-3 accumulation at cell-cell contacts by TROP2 depletion. Confluent undifferentiated WT or TROP2 KO HaCaT cells were treated with Narciclasine (ROCK activator) or acid medium (pH 6.0) and stained to quantify junctional enrichment of claudin-3 (N=5; error bar: ± SD; Tukey-Kramer One-way Anova). ( I ) Schematic illustrating the mechanism of ectopic TJ formation through RhoA activation. In wild-type keratinocytes, claudin and TROP2 form a complex in the Golgi apparatus and are subsequently transported to the plasma membrane. In contrast, in cells lacking TROP2, claudin, which cannot form a complex with TROP2, accumulates in the Golgi apparatus, leading to a significant decrease in the amount of claudin at the plasma membrane. In wild-type cells, TROP2 is cleaved by matriptase activated via the Rho-ROCK pathway. This cleavage results in the breakdown of the TROP2-claudin complex, allowing released claudins to form de novo tight junctions (TJs). Figure 6—source data 1. Full blot data for the western blots shown in . Figure 6—source data 2. Full blot original images for the western blots shown in .

Article Snippet: The following primary antibodies were used for immunofluorescence microscopy and immunoblotting: rabbit anti-claudin-3 pAb (34–1700; Thermo Fisher Scientific); mouse anti-claudin-4 mAb (32–9400; Thermo Fisher Scientific); and rabbit anti-claudin-7 pAb (34–9100; Thermo Fisher Scientific); rabbit anti-α-catenin pAb (C2081; Sigma Aldrich); rabbit anti-claudin-1 pAb (SAB4200534; Sigma Aldrich); rat anti-E-cadherin mAb (ECCD2; Takara Bio); rabbit anti-RhoA mAb (2117; Cell Signaling Technology); rabbit anti-phospho-Myosin Light Chain 2 (Thr18/Thr19) mAb (95777; Cell Signaling Technology); rabbit anti-TROP2 mAb (ab214488; abcam); rabbit anti-EpCAM pAb (ab71916; abcam); mouse anti-GM130 mAb (610822; BD).

Techniques: Molecular Weight, Expressing, Western Blot, Staining, Marker, Cell Culture, Incubation, Activation Assay, Clinical Proteomics, Membrane

Journal: eLife

Article Title: Rho-ROCK liberates sequestered claudin for rapid de novo tight junction formation

doi: 10.7554/eLife.102794

Figure Lengend Snippet: ( A ) Wild-type (WT) HaCaT cells were cultured in Ca +JNK inh medium for indicated time points and time-course change of indicated proteins was examined by western blotting. (N=5; error bar: ± SD; Kruskal-Wallis test followed by Steel-Dwass post hoc test; *p<0.05). ( B ) Quantification of the cleaved TROP2 fragment relative to total TROP2. (N=5; error bar: ± SD; Kruskal-Wallis test followed by Steel-Dwass post hoc test; *p<0.05). ( C , D , and E ) HaCaT cells were cultured in normal medium or Ca +JNK inh medium for 24 hr, fixed, and then stained with the m24 mAb (total matriptase: magenta) and anti-claudin-3 pAb (green; C ) or with the M69 mAb (activated matriptase: magenta) and anti-claudin-3 pAb (green; D ). Scale bar = 20 μm. ( E ) A bar graph illustrating the junctional enrichment of activated matriptase (control: N=3, Ca +JNK inh: N=4; error bar: ± SD; Student’s t-test). ( F ) HaCaT cells were cultured in normal medium or Ca +JNK inh medium supplemented with DMSO (control) or Camostat (serine protease inhibitor) for 24 hr, fixed, and then stained with anti-claudin-3 pAb (green), anti-α-catenin mAb (α–18; magenta). Scale bar: 20 μm. ( G ) WT HaCaT cells and cells over-expressing Hai1-GFP were co-cultured in Ca +JNK inh medium for 24 hr, fixed, and then stained with anti-claudin-1 pAb. Scale bar: 20 μm. ( H ) Bar graph illustrating the effects of either Camostat treatment or over-expression of Hai1 on accumulation of claudin-3 at cell-cell contacts in HaCaT cells cultured in Ca +JNK inh medium (N=6; error bar: ± SD; Tukey-Kramer One-way Anova). ( I ) Mouse ear whole-mount immunofluorescence analysis for TROP2 and ZO-1. The asterisk indicates the nucleus of cells belonging to the indicated layer. ( J ) Schematic illustrating the mechanism of TJ formation during differentiation into the granular layer in the epidermis. In undifferentiated cells, claudin is expressed and bound to TROP2 on the plasma membrane. As cells differentiate into the granular layer, RhoA becomes activated, followed by the activation of matriptase, leading to the cleavage of TROP2. The cleavage of TROP2 results in the dissociation of claudin, and the dissociated claudins then polymerize into de novo TJs. Figure 7—source data 1. Full blot data for the western blots shown in . Figure 7—source data 2. Full blot original images for the western blots shown in .

Article Snippet: The following primary antibodies were used for immunofluorescence microscopy and immunoblotting: rabbit anti-claudin-3 pAb (34–1700; Thermo Fisher Scientific); mouse anti-claudin-4 mAb (32–9400; Thermo Fisher Scientific); and rabbit anti-claudin-7 pAb (34–9100; Thermo Fisher Scientific); rabbit anti-α-catenin pAb (C2081; Sigma Aldrich); rabbit anti-claudin-1 pAb (SAB4200534; Sigma Aldrich); rat anti-E-cadherin mAb (ECCD2; Takara Bio); rabbit anti-RhoA mAb (2117; Cell Signaling Technology); rabbit anti-phospho-Myosin Light Chain 2 (Thr18/Thr19) mAb (95777; Cell Signaling Technology); rabbit anti-TROP2 mAb (ab214488; abcam); rabbit anti-EpCAM pAb (ab71916; abcam); mouse anti-GM130 mAb (610822; BD).

Techniques: Cell Culture, Western Blot, Staining, Control, Protease Inhibitor, Expressing, Over Expression, Immunofluorescence, Clinical Proteomics, Membrane, Activation Assay

Journal: The Journal of Cell Biology

Article Title: Adherens junctions influence tight junction formation via changes in membrane lipid composition

doi: 10.1083/jcb.201711042

Figure Lengend Snippet: α-Catenin–KO cells internalize claudins. (A) Phase-contrast images of WT and α-catenin–KO EpH4 cells. (B) Immunoblotting of whole-cell lysates of WT and α-catenin–KO EpH4 cells with the indicated antibodies. (C) WT and α-catenin–KO EpH4 cells were fixed and costained with an anti–claudin-3 pAb and an anti–E-cadherin mAb (left) or with an anti–JAM-A pAb and an antioccludin mAb (right). (D) α-Catenin–KO EpH4 cells stably expressing GFP-tagged mouse α-catenin were fixed and costained with an anti–claudin-3 pAb and an anti–E-cadherin mAb. (E) Immunoblotting of whole-cell lysates of WT EpH4 cells, α-catenin–KO EpH4 cells, and α-catenin–KO EpH4 cells stably expressing GFP-tagged α-catenin (rescue) with the indicated antibodies. Molecular masses are given in kilodaltons. (F) α-Catenin–KO EpH4 cells were fixed and costained with an anti–claudin-3 pAb (green) and an anti-EEA1 mAb (red, top), an anti-LAMP1 mAb (red, middle), or an anti-GM130 mAb (red, bottom). Arrowheads indicate colocalization. (G) α-Catenin–KO EpH4 cells were treated with DMSO (control, top), 10 µg/ml chlorpromazine (middle) for 1 h, or 100 µM dynasore (bottom) for 2 h, fixed, and stained with an anti–claudin-3 pAb. Bars: (A, C, D, and F) 20 µm; (G) 25 µm.

Article Snippet: The following primary antibodies were used for immunofluorescence microscopy and immunoblotting: rabbit anti–claudin-1 (71-7800), rabbit antioccludin (71-1500), rabbit anti–claudin-3 (34-1700), and rabbit anti–JAM-A (36-1700) polyclonal antibodies (pAbs; Thermo Fisher Scientific); mouse antivinculin and mouse anti–α-tubulin mAbs and a rabbit anti–α-catenin pAb (Sigma-Aldrich); mouse anti-GM130, mouse anti-LAMP1, mouse anti-BiP/Grp78, and mouse antinucleoporin p62 mAbs (BD); a mouse anti–desmoglein-2 mAb (Abcam); a rabbit anti–caveolin-1 pAb (Cell Signaling Technology); a rabbit anti–syntaxin-3 pAb (Synaptic Systems); and mouse anti–ZO-1 (T8754), rat antioccludin (MOC37), and rat ECCD-2 mAbs (Takara Bio Inc.).

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

Journal: The Journal of Cell Biology

Article Title: Adherens junctions influence tight junction formation via changes in membrane lipid composition

doi: 10.1083/jcb.201711042

Figure Lengend Snippet: Cholesterol is enriched in the TJ-containing PM fraction. (A) C1L cells were fixed and stained with an anti–claudin-1 pAb. (B) C1L cells were fixed and stained with an anti–claudin-1 pAb (green) and phalloidin (red). Bars, 10 µm. (C) Immunoblot analysis of the PM and IM fractions of C1L cells. Each membrane fraction (5 µg) was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with antibodies against the indicated marker proteins (left). Coomassie brilliant blue (CBB) staining is shown on the right. (D) Positive ion mass spectra of SM species in the PM fractions of L and C1L cells. The SM molecular species corresponding with each peak are indicated. The x and y axes show the total carbon chain length and the number of carbon–carbon double bonds of individual lipid molecular species, respectively. (E) Quantification of the indicated SM species in the PM fractions of L cells and C1L cells. (F) Quantification of the cholesterol-to-phospholipid ratio in the PM fractions of L and C1L cells. (G) Immunoblot analysis of the DRM and non-DRM fractions of WT and α-catenin–KO EpH4 cells using pAbs against the DRM marker proteins claudin-3 and caveolin-1. Results in C, D, and G are representative of three independent experiments. (H) Quantification of the ratio of the claudin-3 level in the DRM fraction to that in the non-DRM fraction in WT and α-catenin–KO EpH4 cells. Error bars show SD calculated based on three independent experiments (Student’s t test, *, P < 0.05).

Article Snippet: The following primary antibodies were used for immunofluorescence microscopy and immunoblotting: rabbit anti–claudin-1 (71-7800), rabbit antioccludin (71-1500), rabbit anti–claudin-3 (34-1700), and rabbit anti–JAM-A (36-1700) polyclonal antibodies (pAbs; Thermo Fisher Scientific); mouse antivinculin and mouse anti–α-tubulin mAbs and a rabbit anti–α-catenin pAb (Sigma-Aldrich); mouse anti-GM130, mouse anti-LAMP1, mouse anti-BiP/Grp78, and mouse antinucleoporin p62 mAbs (BD); a mouse anti–desmoglein-2 mAb (Abcam); a rabbit anti–caveolin-1 pAb (Cell Signaling Technology); a rabbit anti–syntaxin-3 pAb (Synaptic Systems); and mouse anti–ZO-1 (T8754), rat antioccludin (MOC37), and rat ECCD-2 mAbs (Takara Bio Inc.).

Techniques: Staining, Western Blot, Membrane, SDS Page, Marker

Journal: The Journal of Cell Biology

Article Title: Adherens junctions influence tight junction formation via changes in membrane lipid composition

doi: 10.1083/jcb.201711042

Figure Lengend Snippet: Depletion of cholesterol specifically impairs the formation of TJs. (A) WT EpH4 cells were cultured in transwell chambers and treated with PBS (control) or 75 mM MβCD for the indicated duration, and then cells underwent TER analysis (means ± SD; n = 4). (B) WT EpH4 cells were treated with PBS (control), 25 mM MβCD, 50 mM MβCD, or 75 mM MβCD for 30 min, fixed, and costained with an anti–claudin-3 pAb and an anti–E-cadherin mAb. (C) WT EpH4 cells were treated with PBS (control) or 75 mM MβCD for 30 min, fixed, and costained with an anti–claudin-3 pAb and an anti–desmoglein-2 mAb. (D) WT EpH4 cells were treated with PBS (control) or 50 mM MβCD for 30 min, fixed, and costained with an anti–E-cadherin mAb and an antioccludin pAb. Bars, 20 µm.

Article Snippet: The following primary antibodies were used for immunofluorescence microscopy and immunoblotting: rabbit anti–claudin-1 (71-7800), rabbit antioccludin (71-1500), rabbit anti–claudin-3 (34-1700), and rabbit anti–JAM-A (36-1700) polyclonal antibodies (pAbs; Thermo Fisher Scientific); mouse antivinculin and mouse anti–α-tubulin mAbs and a rabbit anti–α-catenin pAb (Sigma-Aldrich); mouse anti-GM130, mouse anti-LAMP1, mouse anti-BiP/Grp78, and mouse antinucleoporin p62 mAbs (BD); a mouse anti–desmoglein-2 mAb (Abcam); a rabbit anti–caveolin-1 pAb (Cell Signaling Technology); a rabbit anti–syntaxin-3 pAb (Synaptic Systems); and mouse anti–ZO-1 (T8754), rat antioccludin (MOC37), and rat ECCD-2 mAbs (Takara Bio Inc.).

Techniques: Cell Culture, Control

Journal: The Journal of Cell Biology

Article Title: Adherens junctions influence tight junction formation via changes in membrane lipid composition

doi: 10.1083/jcb.201711042

Figure Lengend Snippet: Addition of cholesterol to the PM induces TJ strand formation in α-catenin–KO cells. (A) Time-lapse imaging of α-catenin–KO EpH4 cells expressing GFP–claudin-3. At time 0, 75 mM cholesterol-saturated MβCD was added to the medium to restore the level of cholesterol in the PM. (B) α-Catenin–KO EpH4 cells were treated with PBS (control) or 75 mM cholesterol-saturated MβCD, fixed, and costained with an anti–claudin-3 pAb (green) and an anti–ZO-1 mAb (red). (C) Quantification of the signal intensity of claudin-3 at cell–cell contact areas in α-catenin–KO EpH4 cells before and after loading of cholesterol in the PM. (D) Quantification of the colocalization of claudin-3 and ZO-1 in α-catenin–KO EpH4 cells before and after loading of cholesterol in the PM. The degree of colocalization between claudin-3 and ZO-1 was calculated using ImageJ FIJI software. The value of Pearson’s coefficient of two signals were quantitated. Error bars show SD calculated based on four independent experiments (Student’s t test, *, P < 0.05). (E) Freeze-fracture EM images of TJ strands in α-catenin–KO EpH4 cells treated with PBS (control, top) or 75 mM cholesterol-saturated MβCD (bottom) for 30 min. (F) α-Catenin–KO EpH4 cells were treated 75 mM cholesterol-saturated MβCD, fixed, and stained with an anti–claudin-3 pAb (green) together with an anti–E-cadherin mAb (red, top) or an antivinculin mAb (red, bottom). (G) α-Catenin–KO EpH4 cells expressing GFP–claudin-3 were treated with 75 mM cholesterol-saturated MβCD, fixed with 4% paraformaldehyde, and stained with 50 µg/ml filipin prepared in PBS. (H) α-Catenin–KO EpH4 cells expressing GFP–claudin-3 were treated with DMSO (control, top) or 100 µM dynasore (bottom), fixed with 4% paraformaldehyde, and stained with 50 µg/ml filipin prepared in PBS. (I) Immunoblotting of whole-cell lysates of WT and E-cadherin–KO EpH4 cells with the indicated antibodies. (J) WT and E-cadherin–KO EpH4 cells were fixed with 4% paraformaldehyde and stained with 50 µg/ml filipin prepared in PBS. (K) E-cadherin–KO EpH4 cells were treated with PBS (control) or 75 mM cholesterol-saturated MβCD, fixed, and stained with an anti–claudin-3 pAb. Bars: (A, B, F–H, J, and K) 20 µm; (E) 200 nm.

Article Snippet: The following primary antibodies were used for immunofluorescence microscopy and immunoblotting: rabbit anti–claudin-1 (71-7800), rabbit antioccludin (71-1500), rabbit anti–claudin-3 (34-1700), and rabbit anti–JAM-A (36-1700) polyclonal antibodies (pAbs; Thermo Fisher Scientific); mouse antivinculin and mouse anti–α-tubulin mAbs and a rabbit anti–α-catenin pAb (Sigma-Aldrich); mouse anti-GM130, mouse anti-LAMP1, mouse anti-BiP/Grp78, and mouse antinucleoporin p62 mAbs (BD); a mouse anti–desmoglein-2 mAb (Abcam); a rabbit anti–caveolin-1 pAb (Cell Signaling Technology); a rabbit anti–syntaxin-3 pAb (Synaptic Systems); and mouse anti–ZO-1 (T8754), rat antioccludin (MOC37), and rat ECCD-2 mAbs (Takara Bio Inc.).

Techniques: Imaging, Expressing, Control, Software, Staining, Western Blot