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A Transwell migration assay for quantifying MSCs migration in response to TGF-β. MSCs were treated with TGF-β (10 ng/ml) for 12 h. TGF-β type I receptor inhibitor, SB505124 (SB50, 500 nM) was pretreated for 30 min. The percentage of migrated cell was quantified. Representative images are shown in Supplementary Fig. S ( n = 4 independent cultures). B Sphere migration assay for quantifying MSCs migration in response to TGF-β. MSCs spheres were treated with TGF-β (1 ng/ml) for 24 h. White dashed boxes in a and b are magnified in a’ and b’ , respectively ( n = 2 independent cultures). C Western blot analysis of N-cadherin, α-catenin, and ZO-1. MSCs were pretreated with SB505124 (SB50, 500 nM) followed by the treatment of TGF-β (1 ng/ml) for 48 h. α-Tubulin was used as an internal control ( n = 3 independent cultures). D Expression level of N-cadherin ( CDH2 ), α-catenin ( CTNNA1 ), and ZO-1 ( <t>TJP1</t> ) mRNA in MSCs pretreated with SB505124 (SB50, 500 nM) followed by the treatment of TGF-β (1 ng/ml) for 24 h ( n = 3 independent cultures for N-cadherin, n = 2 independent cultures for α-catenin or ZO-1). E Immunostaining of N-cadherin, α-catenin, and ZO-1 in MSCs pretreated with SB505124 (SB50, 500 nM) followed by TGF-β (1 ng/ml) for 24 h. Actin and nuclei were stained with phalloidin (cyan blue) and DAPI (blue), respectively. RGB profiling was performed using ImageJ in the merged channels of ZO-1 and α-catenin. The analyzed regions are delineated by white lines ( n = 3 independent cultures). F , G Transwell migration assay for quantifying the migration of α-catenin- ( F ) or ZO-1- ( G ) knockdown MSCs in response to TGF-β (10 ng/ml) for 12 h. Representative images are shown in Supplementary Fig. , K ( n = 2 independent cultures for ( F ) and n = 4 independent cultures for ( G )). Scale bar, 100 μm. Results are presented as mean ± SD. P value measured by unpaired Student’s t -test; ** P < 0.01, *** P < 0.001, **** P < 0.0001.
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A Transwell migration assay for quantifying MSCs migration in response to TGF-β. MSCs were treated with TGF-β (10 ng/ml) for 12 h. TGF-β type I receptor inhibitor, SB505124 (SB50, 500 nM) was pretreated for 30 min. The percentage of migrated cell was quantified. Representative images are shown in Supplementary Fig. S ( n = 4 independent cultures). B Sphere migration assay for quantifying MSCs migration in response to TGF-β. MSCs spheres were treated with TGF-β (1 ng/ml) for 24 h. White dashed boxes in a and b are magnified in a’ and b’ , respectively ( n = 2 independent cultures). C Western blot analysis of N-cadherin, α-catenin, and ZO-1. MSCs were pretreated with SB505124 (SB50, 500 nM) followed by the treatment of TGF-β (1 ng/ml) for 48 h. α-Tubulin was used as an internal control ( n = 3 independent cultures). D Expression level of N-cadherin ( CDH2 ), α-catenin ( CTNNA1 ), and ZO-1 ( <t>TJP1</t> ) mRNA in MSCs pretreated with SB505124 (SB50, 500 nM) followed by the treatment of TGF-β (1 ng/ml) for 24 h ( n = 3 independent cultures for N-cadherin, n = 2 independent cultures for α-catenin or ZO-1). E Immunostaining of N-cadherin, α-catenin, and ZO-1 in MSCs pretreated with SB505124 (SB50, 500 nM) followed by TGF-β (1 ng/ml) for 24 h. Actin and nuclei were stained with phalloidin (cyan blue) and DAPI (blue), respectively. RGB profiling was performed using ImageJ in the merged channels of ZO-1 and α-catenin. The analyzed regions are delineated by white lines ( n = 3 independent cultures). F , G Transwell migration assay for quantifying the migration of α-catenin- ( F ) or ZO-1- ( G ) knockdown MSCs in response to TGF-β (10 ng/ml) for 12 h. Representative images are shown in Supplementary Fig. , K ( n = 2 independent cultures for ( F ) and n = 4 independent cultures for ( G )). Scale bar, 100 μm. Results are presented as mean ± SD. P value measured by unpaired Student’s t -test; ** P < 0.01, *** P < 0.001, **** P < 0.0001.
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A Transwell migration assay for quantifying MSCs migration in response to TGF-β. MSCs were treated with TGF-β (10 ng/ml) for 12 h. TGF-β type I receptor inhibitor, SB505124 (SB50, 500 nM) was pretreated for 30 min. The percentage of migrated cell was quantified. Representative images are shown in Supplementary Fig. S ( n = 4 independent cultures). B Sphere migration assay for quantifying MSCs migration in response to TGF-β. MSCs spheres were treated with TGF-β (1 ng/ml) for 24 h. White dashed boxes in a and b are magnified in a’ and b’ , respectively ( n = 2 independent cultures). C Western blot analysis of N-cadherin, α-catenin, and ZO-1. MSCs were pretreated with SB505124 (SB50, 500 nM) followed by the treatment of TGF-β (1 ng/ml) for 48 h. α-Tubulin was used as an internal control ( n = 3 independent cultures). D Expression level of N-cadherin ( CDH2 ), α-catenin ( CTNNA1 ), and ZO-1 ( <t>TJP1</t> ) mRNA in MSCs pretreated with SB505124 (SB50, 500 nM) followed by the treatment of TGF-β (1 ng/ml) for 24 h ( n = 3 independent cultures for N-cadherin, n = 2 independent cultures for α-catenin or ZO-1). E Immunostaining of N-cadherin, α-catenin, and ZO-1 in MSCs pretreated with SB505124 (SB50, 500 nM) followed by TGF-β (1 ng/ml) for 24 h. Actin and nuclei were stained with phalloidin (cyan blue) and DAPI (blue), respectively. RGB profiling was performed using ImageJ in the merged channels of ZO-1 and α-catenin. The analyzed regions are delineated by white lines ( n = 3 independent cultures). F , G Transwell migration assay for quantifying the migration of α-catenin- ( F ) or ZO-1- ( G ) knockdown MSCs in response to TGF-β (10 ng/ml) for 12 h. Representative images are shown in Supplementary Fig. , K ( n = 2 independent cultures for ( F ) and n = 4 independent cultures for ( G )). Scale bar, 100 μm. Results are presented as mean ± SD. P value measured by unpaired Student’s t -test; ** P < 0.01, *** P < 0.001, **** P < 0.0001.
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A Transwell migration assay for quantifying MSCs migration in response to TGF-β. MSCs were treated with TGF-β (10 ng/ml) for 12 h. TGF-β type I receptor inhibitor, SB505124 (SB50, 500 nM) was pretreated for 30 min. The percentage of migrated cell was quantified. Representative images are shown in Supplementary Fig. S ( n = 4 independent cultures). B Sphere migration assay for quantifying MSCs migration in response to TGF-β. MSCs spheres were treated with TGF-β (1 ng/ml) for 24 h. White dashed boxes in a and b are magnified in a’ and b’ , respectively ( n = 2 independent cultures). C Western blot analysis of N-cadherin, α-catenin, and ZO-1. MSCs were pretreated with SB505124 (SB50, 500 nM) followed by the treatment of TGF-β (1 ng/ml) for 48 h. α-Tubulin was used as an internal control ( n = 3 independent cultures). D Expression level of N-cadherin ( CDH2 ), α-catenin ( CTNNA1 ), and ZO-1 ( <t>TJP1</t> ) mRNA in MSCs pretreated with SB505124 (SB50, 500 nM) followed by the treatment of TGF-β (1 ng/ml) for 24 h ( n = 3 independent cultures for N-cadherin, n = 2 independent cultures for α-catenin or ZO-1). E Immunostaining of N-cadherin, α-catenin, and ZO-1 in MSCs pretreated with SB505124 (SB50, 500 nM) followed by TGF-β (1 ng/ml) for 24 h. Actin and nuclei were stained with phalloidin (cyan blue) and DAPI (blue), respectively. RGB profiling was performed using ImageJ in the merged channels of ZO-1 and α-catenin. The analyzed regions are delineated by white lines ( n = 3 independent cultures). F , G Transwell migration assay for quantifying the migration of α-catenin- ( F ) or ZO-1- ( G ) knockdown MSCs in response to TGF-β (10 ng/ml) for 12 h. Representative images are shown in Supplementary Fig. , K ( n = 2 independent cultures for ( F ) and n = 4 independent cultures for ( G )). Scale bar, 100 μm. Results are presented as mean ± SD. P value measured by unpaired Student’s t -test; ** P < 0.01, *** P < 0.001, **** P < 0.0001.
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A Transwell migration assay for quantifying MSCs migration in response to TGF-β. MSCs were treated with TGF-β (10 ng/ml) for 12 h. TGF-β type I receptor inhibitor, SB505124 (SB50, 500 nM) was pretreated for 30 min. The percentage of migrated cell was quantified. Representative images are shown in Supplementary Fig. S ( n = 4 independent cultures). B Sphere migration assay for quantifying MSCs migration in response to TGF-β. MSCs spheres were treated with TGF-β (1 ng/ml) for 24 h. White dashed boxes in a and b are magnified in a’ and b’ , respectively ( n = 2 independent cultures). C Western blot analysis of N-cadherin, α-catenin, and ZO-1. MSCs were pretreated with SB505124 (SB50, 500 nM) followed by the treatment of TGF-β (1 ng/ml) for 48 h. α-Tubulin was used as an internal control ( n = 3 independent cultures). D Expression level of N-cadherin ( CDH2 ), α-catenin ( CTNNA1 ), and ZO-1 ( TJP1 ) mRNA in MSCs pretreated with SB505124 (SB50, 500 nM) followed by the treatment of TGF-β (1 ng/ml) for 24 h ( n = 3 independent cultures for N-cadherin, n = 2 independent cultures for α-catenin or ZO-1). E Immunostaining of N-cadherin, α-catenin, and ZO-1 in MSCs pretreated with SB505124 (SB50, 500 nM) followed by TGF-β (1 ng/ml) for 24 h. Actin and nuclei were stained with phalloidin (cyan blue) and DAPI (blue), respectively. RGB profiling was performed using ImageJ in the merged channels of ZO-1 and α-catenin. The analyzed regions are delineated by white lines ( n = 3 independent cultures). F , G Transwell migration assay for quantifying the migration of α-catenin- ( F ) or ZO-1- ( G ) knockdown MSCs in response to TGF-β (10 ng/ml) for 12 h. Representative images are shown in Supplementary Fig. , K ( n = 2 independent cultures for ( F ) and n = 4 independent cultures for ( G )). Scale bar, 100 μm. Results are presented as mean ± SD. P value measured by unpaired Student’s t -test; ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Journal: Cell Death Discovery

Article Title: ZO-1 regulates the migration of mesenchymal stem cells in cooperation with α-catenin in response to breast tumor cells

doi: 10.1038/s41420-023-01793-4

Figure Lengend Snippet: A Transwell migration assay for quantifying MSCs migration in response to TGF-β. MSCs were treated with TGF-β (10 ng/ml) for 12 h. TGF-β type I receptor inhibitor, SB505124 (SB50, 500 nM) was pretreated for 30 min. The percentage of migrated cell was quantified. Representative images are shown in Supplementary Fig. S ( n = 4 independent cultures). B Sphere migration assay for quantifying MSCs migration in response to TGF-β. MSCs spheres were treated with TGF-β (1 ng/ml) for 24 h. White dashed boxes in a and b are magnified in a’ and b’ , respectively ( n = 2 independent cultures). C Western blot analysis of N-cadherin, α-catenin, and ZO-1. MSCs were pretreated with SB505124 (SB50, 500 nM) followed by the treatment of TGF-β (1 ng/ml) for 48 h. α-Tubulin was used as an internal control ( n = 3 independent cultures). D Expression level of N-cadherin ( CDH2 ), α-catenin ( CTNNA1 ), and ZO-1 ( TJP1 ) mRNA in MSCs pretreated with SB505124 (SB50, 500 nM) followed by the treatment of TGF-β (1 ng/ml) for 24 h ( n = 3 independent cultures for N-cadherin, n = 2 independent cultures for α-catenin or ZO-1). E Immunostaining of N-cadherin, α-catenin, and ZO-1 in MSCs pretreated with SB505124 (SB50, 500 nM) followed by TGF-β (1 ng/ml) for 24 h. Actin and nuclei were stained with phalloidin (cyan blue) and DAPI (blue), respectively. RGB profiling was performed using ImageJ in the merged channels of ZO-1 and α-catenin. The analyzed regions are delineated by white lines ( n = 3 independent cultures). F , G Transwell migration assay for quantifying the migration of α-catenin- ( F ) or ZO-1- ( G ) knockdown MSCs in response to TGF-β (10 ng/ml) for 12 h. Representative images are shown in Supplementary Fig. , K ( n = 2 independent cultures for ( F ) and n = 4 independent cultures for ( G )). Scale bar, 100 μm. Results are presented as mean ± SD. P value measured by unpaired Student’s t -test; ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: To prepare the ZO-1 expressing vector (pLL-CMV-ZO-1-Myc), the full-length human ZO-1 tagged with Myc was amplified from pCB6 ZO1 (addgene #30317, Watertown, MA, USA) using the sense primer 5′- tcggatccaggcctcccgccaccatgTCCGCCAGAGCTGCGGCCGC-3′ which is containing complementary sequences flanking the 5′ upstream SmaI cutting point on pLL-CMV-GFP and the pair region of N-terminus of ZO-1.

Techniques: Transwell Migration Assay, Migration, Western Blot, Control, Expressing, Immunostaining, Staining, Knockdown