Journal: Scientific Reports

Article Title: Nectin-4 and p95-ErbB2 cooperatively regulate Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation

doi: 10.1038/s41598-021-86437-2

Figure Lengend Snippet: Enhancement of T47D cell proliferation by only the combination of nectin-4 and p95-ErbB2, but not by that of nectin-4 and either ErbB2 or ErbB2∆Ex16, in a suspension culture. ( a , b ) Enhancement of T47D cell proliferation by only the combination of nectin-4 and p95-ErbB2, but not by that of nectin-4 and either ErbB2 or ErbB2∆Ex16, in a suspension culture. The T47D cells stably expressing GFP-tagged ErbB2 or each of its splice variants with or without FLAG-tagged nectin-4 (FLAG-Nectin-4) were detached using Accutase. The cells were collected, their numbers were counted, and the same numbers of the cells were seeded in the serum-free medium with supplements on ultra-low attachment 6 well dish. After the incubation for 28 days, the cells were collected and transferred to ultra-low attachment 96 well dish for image acquisition. Then, the cells were re-collected and subjected to quantitative analysis by cell counting as shown in ( b ). The displayed images were acquired using a BZ-X710 microscope and its software BZ-X Analyzer ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-x710/ ) with BZ-H3A Advanced Application software ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-h3a/ ) for image connection. ( c ) Amounts of nectin-4, ErbB2, and its splice variant proteins. The T47D cells stably expressing GFP-tagged ErbB2 or each of its splice variants with or without FLAG-Nectin-4 were cultured for 72 h in an adherent culture. The cells were subjected to Western blotting using the indicated antibodies (Abs). All the cell lines used in the experiments were the bulk of collected cells and not singly picked-up clones. Arrowhead and square brackets indicate each of the proteins. The displayed blots were cropped, and the full-length blots are shown in Supplementary Figure . Bars indicate the means ± S.D. of five independent experiments and the actual P values for each test are shown. Scale bars 2000 μm or 200 μm (insets). IB, immunoblotting. Representative results (images) from three independent experiments were shown.

Article Snippet: The displayed images were acquired using a BZ-X710 microscope and its software BZ-X Analyzer ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-x710/ ) with BZ-H3A Advanced Application software ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-h3a/ ) for image connection. ( c ) Decrease in the amount of the SOX2 protein by SOX2 knockdown.

Techniques: Stable Transfection, Expressing, Incubation, Cell Counting, Microscopy, Software, Fluorescence, Variant Assay, Cell Culture, Western Blot, Clone Assay

Journal: Scientific Reports

Article Title: Nectin-4 and p95-ErbB2 cooperatively regulate Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation

doi: 10.1038/s41598-021-86437-2

Figure Lengend Snippet: PI3K-AKT signaling- and the Ras-Raf-MEK-ERK signaling-dependent, but not the JAK-STAT signaling-dependent, enhancement of T47D cell proliferation by nectin-4 and p95-ErbB2 in a suspension culture. ( a , b ) The PI3K-AKT signaling- and the Ras-Raf-MEK-ERK signaling-dependent, but not the JAK-STAT signaling-dependent, enhancement of T47D cell proliferation by nectin-4 and p95-ErbB2 in a suspension culture. The T47D cells stably expressing GFP-tagged p95-ErbB2 (p95-ErbB2-GFP) with FLAG-tagged nectin-4 (FLAG-Nectin-4) were detached using Accutase. The cells were collected, their numbers were counted, and the same numbers of the cells were seeded in the serum-free medium with supplements on ultra-low attachment 6 well dish. After the incubation with the PI3K inhibitor LY294002 at 50 μM, the MEK inhibitor U0126 at 10 μM, or the JAK1/2 inhibitor ruxolitinib at 1 μM for 28 days, the cells were collected and transferred to ultra-low attachment 96 well dish for image acquisition. Then, the cells were re-collected and subjected to quantitative analysis by cell counting as shown in ( b ). The displayed images were acquired using a BZ-X710 microscope and its software BZ-X Analyzer ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-x710/ ) with BZ-H3A Advanced Application software ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-h3a/ ) for image connection. The T47D cells stably expressing p95-ErbB2-GFP with FLAG-Nectin-4 used in the experiments were the bulk of collected cells and not singly picked-up clones. Bars indicate the means ± S.D. of three independent experiments and the actual P values for each test are shown. Scale bars 2000 μm or 200 μm (insets). Representative results (images) from three independent experiments were shown.

Article Snippet: The displayed images were acquired using a BZ-X710 microscope and its software BZ-X Analyzer ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-x710/ ) with BZ-H3A Advanced Application software ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-h3a/ ) for image connection. ( c ) Decrease in the amount of the SOX2 protein by SOX2 knockdown.

Techniques: Stable Transfection, Expressing, Incubation, Cell Counting, Microscopy, Software, Fluorescence, Clone Assay

Journal: Scientific Reports

Article Title: Nectin-4 and p95-ErbB2 cooperatively regulate Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation

doi: 10.1038/s41598-021-86437-2

Figure Lengend Snippet: PI3K-AKT signaling-dependent increase in the amount of the SOX2 protein by only the combination of nectin-4 and p95-ErbB2, but not by that of nectin-4 and either ErbB2 or ErbB2∆Ex16, in T47D cells cultured in an adherent culture. ( a – c ) Increase in the amount of the SOX2 protein by only the combination of nectin-4 and p95-ErbB2, but not by that of nectin-4 and either ErbB2 or ErbB2ΔEx16, in T47D cells in an adherent culture. The T47D cells stably expressing GFP-tagged ErbB2 or each of its splice variants with or without FLAG-tagged nectin-4 (FLAG-Nectin-4) were cultured for 72 h in an adherent culture. The cells were fixed and stained with the indicated Ab and Hoechst33342 as shown in ( a ). The number of the cells that prominently express SOX2 was counted by fluorescence microscopy as shown in ( b ). The T47D cells stably expressing GFP-tagged ErbB2 or each of its splice variants with or without FLAG-Nectin-4 were cultured for 72 h in an adherent culture. The cells were subjected to Western blotting using the indicated Abs as shown in ( c ). Arrowhead and square brackets indicate each of the proteins. The displayed blots were cropped, and the full-length blots are shown in Supplementary Figure a. ( d – f ) Requirement of the PI3K-AKT signaling-, but not the Ras-Raf-MEK-ERK signaling-dependent or the JAK-STAT signaling-dependent increase in the amount of the SOX2 protein by nectin-4 and p95-ErbB2 in T47D cells in an adherent culture. The T47D cells stably expressing GFP-tagged p95-ErbB2 (p95-ErbB2-GFP) with FLAG-Nectin-4 were cultured for 48 h. The cells were then treated with the PI3K inhibitor LY294002 at 50 μM, the MEK inhibitor U0126 at 10 μM, or the JAK1/2 inhibitor ruxolitinib at 1 μM for 24 h. After the incubation, the assays were carried out as in ( a – c ). The displayed images were acquired using a BZ-X710 microscope and its software BZ-X Analyzer ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-x710/ ) and processed using ImageJ version 1.48v 32-bit software ( https://imagej.nih.gov/ij/ ) for color changes of the images. The displayed blots were cropped, and the full-length blots are shown in Supplementary Figure b. All the cell lines used in the experiments were the bulk of collected cells and not singly picked-up clones. Bars indicate the means ± S.D. of three independent experiments and the actual P values for each test are shown in each figure. Scale bars 50 μm. IB, immunoblotting. Representative results (images) from three independent experiments were shown.

Article Snippet: The displayed images were acquired using a BZ-X710 microscope and its software BZ-X Analyzer ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-x710/ ) with BZ-H3A Advanced Application software ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-h3a/ ) for image connection. ( c ) Decrease in the amount of the SOX2 protein by SOX2 knockdown.

Techniques: Cell Culture, Stable Transfection, Expressing, Staining, Fluorescence, Microscopy, Western Blot, Incubation, Software, Clone Assay

Journal: Scientific Reports

Article Title: Nectin-4 and p95-ErbB2 cooperatively regulate Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation

doi: 10.1038/s41598-021-86437-2

Figure Lengend Snippet: SOX2-dependent enhancement of T47D cell proliferation by nectin-4 and p95-ErbB2 in a suspension culture. ( a , b ) Requirement of SOX2 for the enhancement of T47D cell proliferation by nectin-4 and p95-ErbB2 in a suspension culture. The T47D cells stably expressing GFP-tagged p95-ErbB2 (p95-ErbB2-GFP) with FLAG-tagged nectin-4 (FLAG-Nectin-4) that also stably express control shRNA or SOX2 shRNAs were detached using Accutase. The cells were collected, their numbers were counted, and the same numbers of the cells were seeded in the serum-free medium with supplements on ultra-low attachment 6 well dish. After the incubation for 28 days, the cells were collected and transferred to ultra-low attachment 96 well dish for image acquisition. Then, the cells were re-collected and subjected to quantitative analysis by cell counting as shown in ( b ). The displayed images were acquired using a BZ-X710 microscope and its software BZ-X Analyzer ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-x710/ ) with BZ-H3A Advanced Application software ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-h3a/ ) for image connection. ( c ) Decrease in the amount of the SOX2 protein by SOX2 knockdown. The T47D cells stably expressing p95-ErbB2-GFP with FLAG-Nectin-4 that also stably express control shRNA or SOX2 shRNAs were cultured for 72 h in an adherent culture. The cells were subjected to Western blotting using the indicated Abs. Arrowhead and square bracket indicate each of the proteins. The displayed blots were cropped, and the full-length blots are shown in Supplementary Figure . All the cell lines used in the experiments were the bulk of collected cells and not singly picked-up clones. Bars indicate the means ± S.D. of three independent experiments and the actual P values for each test are shown. Scale bars 2000 μm or 200 μm (insets). IB, immunoblotting. Representative results (images) from three independent experiments were shown.

Article Snippet: The displayed images were acquired using a BZ-X710 microscope and its software BZ-X Analyzer ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-x710/ ) with BZ-H3A Advanced Application software ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-h3a/ ) for image connection. ( c ) Decrease in the amount of the SOX2 protein by SOX2 knockdown.

Techniques: Stable Transfection, Expressing, shRNA, Incubation, Cell Counting, Microscopy, Software, Fluorescence, Cell Culture, Western Blot, Clone Assay

Journal: Scientific Reports

Article Title: Nectin-4 and p95-ErbB2 cooperatively regulate Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation

doi: 10.1038/s41598-021-86437-2

Figure Lengend Snippet: YAP inactivation-dependent enhancement of T47D cell proliferation by nectin-4 and p95-ErbB2 in a suspension culture. ( a , b ) Requirement of the MST1/2 activation for the enhancement of T47D cell proliferation in a suspension culture by nectin-4 and p95-ErbB2. The T47D cells stably expressing GFP-tagged p95-ErbB2 (p95-ErbB2-GFP) with FLAG-tagged nectin-4 (FLAG-Nectin-4) were detached using Accutase. The cells were collected, their numbers were counted, and the same numbers of the cells were seeded in serum-free medium with supplements on ultra-low attachment 6 well dish. After the incubation with the MST1/2 inhibitor XMU-MP-1 at 1 μM for 28 days, the cells were collected and transferred to ultra-low attachment 96 well dish for image acquisition. Then, the cells were re-collected and subjected to quantitative analysis by cell counting as shown in ( b ). The displayed images were acquired using a BZ-X710 microscope and its software BZ-X Analyzer ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-x710/ ) with BZ-H3A Advanced Application software ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-h3a/ ) for image connection. The T47D cells stably expressing p95-ErbB2-GFP with FLAG-Nectin-4 used in the experiments were the bulk of collected cells and not singly picked-up clones. Bars indicate the means ± S.D. of three independent experiments and the actual P value is shown in each figure. Scale bars 2000 μm or 200 μm (insets). Representative results (images) from three independent experiments were shown.

Article Snippet: The displayed images were acquired using a BZ-X710 microscope and its software BZ-X Analyzer ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-x710/ ) with BZ-H3A Advanced Application software ( https://www.keyence.co.jp/products/microscope/fluorescence-microscope/bz-x700/models/bz-h3a/ ) for image connection. ( c ) Decrease in the amount of the SOX2 protein by SOX2 knockdown.

Techniques: Activation Assay, Stable Transfection, Expressing, Incubation, Cell Counting, Microscopy, Software, Fluorescence, Clone Assay