Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 1. Variations in erbb2 expression in developing heart. (A) Schematic diagram illustrating the purification of embryonic hearts, isolation of cardiac cells, and sub- sequent single-cell RNA sequencing analysis. (B) Heatmap showing the expression of enriched markers in cell clusters of zebrafish hearts at 72 hours postfertilization (hpf). CM-V, ventricular cardiomyocytes; FB, fibroblasts; EP, epicardial cells; CM-A, atrial cardiomyocytes; RBC, erythrocytes; VMC, valve myocardial cell; EC, endocardial cells; VEC, valve endocardial cells; IC, immune cells; EPDC, epicardium-derived cells. Yellow, high expression; magenta, low expression. (C) UMAP plot showing the cardiac cell clusters in zebrafish hearts at 72 hpf. (D) UMAP plot showing the heterogeneous expression of erbb2 in CM-V subpopulations. (E) UMAP plot showing reclustering of CM-V. (F) erbb2 heterogeneity in CM-V. Red, high expression; gray, low expression. Three independent experiments were performed. (G to I) Confocal slices of cardiac ventricles expressing erbb2:GFP;myl7:mCherry at (G) 60 hpf, (H) 72 hpf, and (I) 96 hpf. (J) Confocal sections of erbb2:GFP;myl7:mCherry hearts at 45 days postfertilization (dpf). Enlarged views of boxed areas are shown in the right panels. White arrowheads, erbb2:GFP+ cardiomyocytes; magenta arrowheads, erbb2:GFP− cardiomyocytes. CL CMs, compact layer cardiomyocytes; TL CMs, trabecular layer cardiomyocytes. [(G) to (J)] n = 10 zebrafish for each stage. Scale bars, 20 μm [(G) to (I)] and 100 μm (J).

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Expressing, Purification, Isolation, RNA Sequencing, Derivative Assay

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 2. The erbb2 heterogeneity correlates with trabeculae formation. (A) erbb2:RFP;β-act2:BSR embryos were injected with photoactivatable green fluorescent pro- tein (PAGFP) mRNA at the one-cell stage. Time series images of the same embryonic heart at 60 hpf before ultraviolet (UV) photoactivation (top), immediately following photoactivation (middle), and at 80 hpf (bottom). Magenta arrowheads: erbb2:GFP+ cardiomyocytes; white arrowheads: erbb2:GFP− cardiomyocytes. (B) Schematic of photoactivation experiments to track the delamination of single cardiomyocytes in (A). (C) Quantification of the spatial distribution of erbb2+ and erbb2− cardiomyocytes in the trabecular and compact layers at 80 hpf in (A). n = 12 embryos. (D) Schematic diagram showing the dual-side illumination objectives coupled with an imaging module. (E) Time-lapse images of erbb2:GFP;myl7:H2A-mCherry heart between 60 and 80 hpf. Blue and white arrowheads indicate erbb2+ and erbb2− cardiomyocytes, re- spectively. (F) Curved lines in (E) represent the moving trajectories of erbb2+ (magenta) and erbb2− (blue) cardiomyocyte nuclei. Blue circles indicate the starting points, and yellow circles denote the end points. (G) The schematic depicts the distance along the apicobasal axis of cell delamination over a 20-hour time window. (H) Quantifi- cation of the movement distance of erbb2+ and erbb2− cardiomyocytes. n = 12 cardiomyocytes per group. For in toto live imaging, three independent experiments were performed using different hearts on different days. Data are presented as mean ± SEM. Two-tailed Mann-Whitney U tests were used for comparison. Scale bars, 25 μm.

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Injection, Imaging, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 3. erbb2 directly controls tension heterogeneity to guide cardiomyocyte delamination. (A) Left, p-myo staining in erbb2:GFP hearts at 60 hpf. Dashed arrows, the posi- tions subjected to fluorescence intensity profiling; blue and magenta arrowhead, erbb2:GFP+ and erbb2:GFP− cardiomyocytes, respectively. Middle and right: fluorescence in- tensity profiles and average fluorescence intensities of p-myo. n = 12 cardiomyocytes for each group. (B) Apical view and schematics of F-actin maximum intensity projections in erbb2:GFP embryos at 96 hpf. White and magenta dashed lines, erbb2+ and erbb2− cells, respectively. (C) Confocal image of erbb2:GFP;myl7:WT-MYL9-mRuby embryo at 60 hpf. White and magenta arrowheads, erbb2:GFP+ and erbb2:GFP− cardiomyocytes. (B and C) n = 7 embryos. (D) Fluorescence recovery after photobleaching (FRAP) analysis of WT- MYL9-mRuby in erbb2:GFP;myl7:WT-MYL9-mRuby hearts. Right: quantifications of WT-MYL9-mRuby mobile fraction. n = 7 cardiomyocytes for each group. (E) F-actin staining in myl7:memGFP hearts treated with DMSO, PD168393, and AG1478. Arrowheads, the apical enrichment of F-actin. (F) Schematic of a dominant-negative human ERBB2 (DN-ERBB2). (G) myl7:DN-ERBB2-P2A-mCherry clones were confined to the compact layer at 96 hpf. (H) Quantifications of the spatial distributions of DN-ERBB2 clones at 72 and 96 hpf. CL CMs, compact layer cardiomyocytes; TL CMs, trabecular layer cardiomyocytes. [(E), (G), and (H)] n = 8 embryos for each stage. (I) Apical view and schematics of F-actin in WT and myl7:DN-ERBB2-P2A-GFP hearts. (J) Confocal image and statistics of trabeculae in myl7:WT-MYL9-mRuby (left), myl7:CA-MYL9-mRuby treated with DMSO (middle), and PD168393 (right) at 72 hpf. Arrowheads, trabeculae. (K) Confocal image showing DN-ERBB2-P2A-GFP+ clones in myl7:WT-MYL9-mRuby and myl7:CA-MYL9-mRuby hearts. (L) Quantifications of DN-ERBB2 clone distributions in (K). [(I) to (L)] n = 7 to 10 embryos for each group. Data are presented as mean ± SEM. Unpaired two-tailed Student’s t tests [(A) and (D)], one-way ANOVA (J), and two-tailed Mann-Whitney U tests [(H) and (L)] were applied to assess statistical significance. Scale bars, 20 μm.

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Staining, Fluorescence, Dominant Negative Mutation, Clone Assay, Two Tailed Test, MANN-WHITNEY

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 4. erbb2 promotes trabeculation by triggering PI3K-mediated remodeling of the actomyosin network. (A) Confocal imaging of myl7:PH-AKT-GFP;erbb2:RFP hearts at 60 hpf. PH-AKT-GFP was enriched on the plasma membrane of erbb2:RFP+ cardiomyocytes, while its cytosolic distribution was observed in erbb2:RFP− cells. Insets are magnifications of boxed areas, and the dashed arrows indicate the position subjected to fluorescence intensity analysis. Right: fluorescence intensity profiles of PH- AKT-GFP. n = 10 embryos. (B) PD168393-treated myl7:PH-AKT-GFP;erbb2:RFP heart at 60 hpf. (C) Confocal image of myl7:PH-AKT-GFP;myl7:DN-ERBB2-P2A-mCherry heart at 60 hpf. (D) myl7:PH-AKT-GFP;erbb2:RFP hearts treated with LY294002 to inhibit PI3K signaling. [(B) to (D)] n = 8 embryos. (E) myl7:memGFP embryos, carrying myl7:actr2b- mRuby clones, were treated with DMSO (left) and PD168393 (right). Arrowheads denote the membrane localization of the actr2b-mRuby in delaminating cardiomyocytes. (F) Apical views of maximum intensity projections of F-actin in wild-type (WT) and myl7:arpin-mRuby hearts. F-actin is restricted to the lateral sides of cardiomyocytes in myl7:arpin-mRuby hearts. (G) Confocal images of myl7:memGFP (left) and myl7:arpin-mRuby;myl7:memGFP (right) hearts at 84 hpf. Arrowheads, trabeculae. (H) Apical views of F-actin network in DMSO and JLY-treated hearts. JLY decouples PI3K signaling from its ability to regulate actin dynamics. JLY, a combination of jasplakinolide (0.8 μM), latrunculin A (0.125 μM), and Y27632 (1 μM). [(E) to (H)] n = 6 to 10 embryos per group. Insets are enlarged views of boxed areas. [(F) and (H)] Dashed arrows indicate the intensity profile of F-actin. (I) Confocal images of 72 hpf myl7:WT-MYL9-mRuby;myl7:memGFP (left) and myl7:CA-MYL9-mRuby;myl7:memGFP (right) embryos treated with DMSO, LY294002, and CK666. Arrowheads indicate trabeculae. Quantifications of trabeculae are displayed on the right. n = 7 embryos per group. One-way ANOVA, followed by Tukey’s multiple comparisons test was used. Data are presented as mean ± SEM. Scale bars, 20 μm. (J) Schematic diagram showing erbb2 promotes actomyo- sin network remodeling through the PI3K-Arp2/3 axis.

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Imaging, Clinical Proteomics, Membrane, Fluorescence, Clone Assay

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 5. Notch-mediated lateral inhibition suppresses trabeculation by reducing erbb2 expression. myl7:mCherry embryos were treated with 100 μM DAPT and DMSO from 84 to 96 hpf. Three independent replicates were performed, with each replicate containing approximately 1000 embryonic hearts. (A) RNA sequencing analysis re- vealed that erbb2 expression was significantly up-regulated following DAPT-treatment. (B) Gene set enrichment analysis (GSEA) revealed an increased expression level of key genes in the PI3K-AKT signaling pathway in Notch-inhibited hearts. Magenta, high expression; blue, low expression. (C) Confocal section (top) and maximum projec- tions (bottom) of Tp1:d2GFP;erbb2:RFP heart at 96 hpf. Note the mutually exclusive distributions of Tp1:d2GFP+ and erbb2:RFP+ cells. White arrowheads, Tp1:d2GFP+ cells; blue arrowheads, erbb2:RFP+ cells. (D) Maximum projections of Tp1:d2GFP;erbb2:RFP embryos treated with DMSO and DAPT. (E) erbb2:RFP embryos were injected with the myl7:NICD-P2A-GFP plasmid at the one-cell stage and imaged at 72 and 84 hpf. White arrowheads, NICD-P2A-GFP+ cells; blue arrowheads, erbb2:GFP+ cells. (F) Confocal images of myl7:PH-AKT-GFP hearts exposed to DMSO and DAPT. Insets are magnifications of boxed areas. (C to F) n = 10 embryos per group. (G) F-actin staining of erbb2:GFP hearts after treatment with either DMSO or DAPT. (H) Confocal images of myl7:WT-MYL9-mRuby (left) and myl7:DN-MYL9-mRuby (right) embryos treated with DMSO and DAPT. Notably, inhibiting Notch activity was unable to rescue the trabeculation defects in myl7:DN-MYL9-mRuby hearts. (I) Confocal images of myl7:WT-MYL9- mRuby (left), myl7:WT-MYL9-mRuby;myl7:NICD-P2A-GFP (middle), and myl7:CA-MYL9-mRuby;myl7:NICD-P2A-GFP (right) hearts. [(G) to (I)] n = 8 embryos per group. Scale bar, 20 μm. (J) Schematic illustration of the trabeculae formation paradigm: Initially, erbb2 is differentially expressed in the single-layer myocardium (left). Subsequently, erbb2 activates the PI3K-Arp2/3 axis, inducing cell tension heterogeneity and cardiomyocyte sprouting (middle). Last, the nascent trabeculae trigger Notch activity in adjacent cardiomyocytes, which then abolishes their erbb2 expression to confine these cardiomyocytes in the compact layer.

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Inhibition, Expressing, RNA Sequencing, Injection, Plasmid Preparation, Staining, Activity Assay

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 1. Variations in erbb2 expression in developing heart. (A) Schematic diagram illustrating the purification of embryonic hearts, isolation of cardiac cells, and sub- sequent single-cell RNA sequencing analysis. (B) Heatmap showing the expression of enriched markers in cell clusters of zebrafish hearts at 72 hours postfertilization (hpf). CM-V, ventricular cardiomyocytes; FB, fibroblasts; EP, epicardial cells; CM-A, atrial cardiomyocytes; RBC, erythrocytes; VMC, valve myocardial cell; EC, endocardial cells; VEC, valve endocardial cells; IC, immune cells; EPDC, epicardium-derived cells. Yellow, high expression; magenta, low expression. (C) UMAP plot showing the cardiac cell clusters in zebrafish hearts at 72 hpf. (D) UMAP plot showing the heterogeneous expression of erbb2 in CM-V subpopulations. (E) UMAP plot showing reclustering of CM-V. (F) erbb2 heterogeneity in CM-V. Red, high expression; gray, low expression. Three independent experiments were performed. (G to I) Confocal slices of cardiac ventricles expressing erbb2:GFP;myl7:mCherry at (G) 60 hpf, (H) 72 hpf, and (I) 96 hpf. (J) Confocal sections of erbb2:GFP;myl7:mCherry hearts at 45 days postfertilization (dpf). Enlarged views of boxed areas are shown in the right panels. White arrowheads, erbb2:GFP+ cardiomyocytes; magenta arrowheads, erbb2:GFP− cardiomyocytes. CL CMs, compact layer cardiomyocytes; TL CMs, trabecular layer cardiomyocytes. [(G) to (J)] n = 10 zebrafish for each stage. Scale bars, 20 μm [(G) to (I)] and 100 μm (J).

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Expressing, Purification, Isolation, RNA Sequencing, Derivative Assay

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 2. The erbb2 heterogeneity correlates with trabeculae formation. (A) erbb2:RFP;β-act2:BSR embryos were injected with photoactivatable green fluorescent pro- tein (PAGFP) mRNA at the one-cell stage. Time series images of the same embryonic heart at 60 hpf before ultraviolet (UV) photoactivation (top), immediately following photoactivation (middle), and at 80 hpf (bottom). Magenta arrowheads: erbb2:GFP+ cardiomyocytes; white arrowheads: erbb2:GFP− cardiomyocytes. (B) Schematic of photoactivation experiments to track the delamination of single cardiomyocytes in (A). (C) Quantification of the spatial distribution of erbb2+ and erbb2− cardiomyocytes in the trabecular and compact layers at 80 hpf in (A). n = 12 embryos. (D) Schematic diagram showing the dual-side illumination objectives coupled with an imaging module. (E) Time-lapse images of erbb2:GFP;myl7:H2A-mCherry heart between 60 and 80 hpf. Blue and white arrowheads indicate erbb2+ and erbb2− cardiomyocytes, re- spectively. (F) Curved lines in (E) represent the moving trajectories of erbb2+ (magenta) and erbb2− (blue) cardiomyocyte nuclei. Blue circles indicate the starting points, and yellow circles denote the end points. (G) The schematic depicts the distance along the apicobasal axis of cell delamination over a 20-hour time window. (H) Quantifi- cation of the movement distance of erbb2+ and erbb2− cardiomyocytes. n = 12 cardiomyocytes per group. For in toto live imaging, three independent experiments were performed using different hearts on different days. Data are presented as mean ± SEM. Two-tailed Mann-Whitney U tests were used for comparison. Scale bars, 25 μm.

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Injection, Imaging, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 3. erbb2 directly controls tension heterogeneity to guide cardiomyocyte delamination. (A) Left, p-myo staining in erbb2:GFP hearts at 60 hpf. Dashed arrows, the posi- tions subjected to fluorescence intensity profiling; blue and magenta arrowhead, erbb2:GFP+ and erbb2:GFP− cardiomyocytes, respectively. Middle and right: fluorescence in- tensity profiles and average fluorescence intensities of p-myo. n = 12 cardiomyocytes for each group. (B) Apical view and schematics of F-actin maximum intensity projections in erbb2:GFP embryos at 96 hpf. White and magenta dashed lines, erbb2+ and erbb2− cells, respectively. (C) Confocal image of erbb2:GFP;myl7:WT-MYL9-mRuby embryo at 60 hpf. White and magenta arrowheads, erbb2:GFP+ and erbb2:GFP− cardiomyocytes. (B and C) n = 7 embryos. (D) Fluorescence recovery after photobleaching (FRAP) analysis of WT- MYL9-mRuby in erbb2:GFP;myl7:WT-MYL9-mRuby hearts. Right: quantifications of WT-MYL9-mRuby mobile fraction. n = 7 cardiomyocytes for each group. (E) F-actin staining in myl7:memGFP hearts treated with DMSO, PD168393, and AG1478. Arrowheads, the apical enrichment of F-actin. (F) Schematic of a dominant-negative human ERBB2 (DN-ERBB2). (G) myl7:DN-ERBB2-P2A-mCherry clones were confined to the compact layer at 96 hpf. (H) Quantifications of the spatial distributions of DN-ERBB2 clones at 72 and 96 hpf. CL CMs, compact layer cardiomyocytes; TL CMs, trabecular layer cardiomyocytes. [(E), (G), and (H)] n = 8 embryos for each stage. (I) Apical view and schematics of F-actin in WT and myl7:DN-ERBB2-P2A-GFP hearts. (J) Confocal image and statistics of trabeculae in myl7:WT-MYL9-mRuby (left), myl7:CA-MYL9-mRuby treated with DMSO (middle), and PD168393 (right) at 72 hpf. Arrowheads, trabeculae. (K) Confocal image showing DN-ERBB2-P2A-GFP+ clones in myl7:WT-MYL9-mRuby and myl7:CA-MYL9-mRuby hearts. (L) Quantifications of DN-ERBB2 clone distributions in (K). [(I) to (L)] n = 7 to 10 embryos for each group. Data are presented as mean ± SEM. Unpaired two-tailed Student’s t tests [(A) and (D)], one-way ANOVA (J), and two-tailed Mann-Whitney U tests [(H) and (L)] were applied to assess statistical significance. Scale bars, 20 μm.

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Staining, Fluorescence, Dominant Negative Mutation, Clone Assay, Two Tailed Test, MANN-WHITNEY

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 4. erbb2 promotes trabeculation by triggering PI3K-mediated remodeling of the actomyosin network. (A) Confocal imaging of myl7:PH-AKT-GFP;erbb2:RFP hearts at 60 hpf. PH-AKT-GFP was enriched on the plasma membrane of erbb2:RFP+ cardiomyocytes, while its cytosolic distribution was observed in erbb2:RFP− cells. Insets are magnifications of boxed areas, and the dashed arrows indicate the position subjected to fluorescence intensity analysis. Right: fluorescence intensity profiles of PH- AKT-GFP. n = 10 embryos. (B) PD168393-treated myl7:PH-AKT-GFP;erbb2:RFP heart at 60 hpf. (C) Confocal image of myl7:PH-AKT-GFP;myl7:DN-ERBB2-P2A-mCherry heart at 60 hpf. (D) myl7:PH-AKT-GFP;erbb2:RFP hearts treated with LY294002 to inhibit PI3K signaling. [(B) to (D)] n = 8 embryos. (E) myl7:memGFP embryos, carrying myl7:actr2b- mRuby clones, were treated with DMSO (left) and PD168393 (right). Arrowheads denote the membrane localization of the actr2b-mRuby in delaminating cardiomyocytes. (F) Apical views of maximum intensity projections of F-actin in wild-type (WT) and myl7:arpin-mRuby hearts. F-actin is restricted to the lateral sides of cardiomyocytes in myl7:arpin-mRuby hearts. (G) Confocal images of myl7:memGFP (left) and myl7:arpin-mRuby;myl7:memGFP (right) hearts at 84 hpf. Arrowheads, trabeculae. (H) Apical views of F-actin network in DMSO and JLY-treated hearts. JLY decouples PI3K signaling from its ability to regulate actin dynamics. JLY, a combination of jasplakinolide (0.8 μM), latrunculin A (0.125 μM), and Y27632 (1 μM). [(E) to (H)] n = 6 to 10 embryos per group. Insets are enlarged views of boxed areas. [(F) and (H)] Dashed arrows indicate the intensity profile of F-actin. (I) Confocal images of 72 hpf myl7:WT-MYL9-mRuby;myl7:memGFP (left) and myl7:CA-MYL9-mRuby;myl7:memGFP (right) embryos treated with DMSO, LY294002, and CK666. Arrowheads indicate trabeculae. Quantifications of trabeculae are displayed on the right. n = 7 embryos per group. One-way ANOVA, followed by Tukey’s multiple comparisons test was used. Data are presented as mean ± SEM. Scale bars, 20 μm. (J) Schematic diagram showing erbb2 promotes actomyo- sin network remodeling through the PI3K-Arp2/3 axis.

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Imaging, Clinical Proteomics, Membrane, Fluorescence, Clone Assay

Journal: Science advances

Article Title: Genetically encoded tension heterogeneity sculpts cardiac trabeculation.

doi: 10.1126/sciadv.ads2998

Figure Lengend Snippet: Fig. 5. Notch-mediated lateral inhibition suppresses trabeculation by reducing erbb2 expression. myl7:mCherry embryos were treated with 100 μM DAPT and DMSO from 84 to 96 hpf. Three independent replicates were performed, with each replicate containing approximately 1000 embryonic hearts. (A) RNA sequencing analysis re- vealed that erbb2 expression was significantly up-regulated following DAPT-treatment. (B) Gene set enrichment analysis (GSEA) revealed an increased expression level of key genes in the PI3K-AKT signaling pathway in Notch-inhibited hearts. Magenta, high expression; blue, low expression. (C) Confocal section (top) and maximum projec- tions (bottom) of Tp1:d2GFP;erbb2:RFP heart at 96 hpf. Note the mutually exclusive distributions of Tp1:d2GFP+ and erbb2:RFP+ cells. White arrowheads, Tp1:d2GFP+ cells; blue arrowheads, erbb2:RFP+ cells. (D) Maximum projections of Tp1:d2GFP;erbb2:RFP embryos treated with DMSO and DAPT. (E) erbb2:RFP embryos were injected with the myl7:NICD-P2A-GFP plasmid at the one-cell stage and imaged at 72 and 84 hpf. White arrowheads, NICD-P2A-GFP+ cells; blue arrowheads, erbb2:GFP+ cells. (F) Confocal images of myl7:PH-AKT-GFP hearts exposed to DMSO and DAPT. Insets are magnifications of boxed areas. (C to F) n = 10 embryos per group. (G) F-actin staining of erbb2:GFP hearts after treatment with either DMSO or DAPT. (H) Confocal images of myl7:WT-MYL9-mRuby (left) and myl7:DN-MYL9-mRuby (right) embryos treated with DMSO and DAPT. Notably, inhibiting Notch activity was unable to rescue the trabeculation defects in myl7:DN-MYL9-mRuby hearts. (I) Confocal images of myl7:WT-MYL9- mRuby (left), myl7:WT-MYL9-mRuby;myl7:NICD-P2A-GFP (middle), and myl7:CA-MYL9-mRuby;myl7:NICD-P2A-GFP (right) hearts. [(G) to (I)] n = 8 embryos per group. Scale bar, 20 μm. (J) Schematic illustration of the trabeculae formation paradigm: Initially, erbb2 is differentially expressed in the single-layer myocardium (left). Subsequently, erbb2 activates the PI3K-Arp2/3 axis, inducing cell tension heterogeneity and cardiomyocyte sprouting (middle). Last, the nascent trabeculae trigger Notch activity in adjacent cardiomyocytes, which then abolishes their erbb2 expression to confine these cardiomyocytes in the compact layer.

Article Snippet: DN- ERBB2 (65224), CA- ERBB2 (16259), and PH- ATK- GFP (51465) plasmids were obtained from Addgene.

Techniques: Inhibition, Expressing, RNA Sequencing, Injection, Plasmid Preparation, Staining, Activity Assay

Journal: The Journal of investigative dermatology

Article Title: Nuclear IL-33 Plays an Important Role in IL-31‒Mediated Downregulation of FLG, Keratin 1, and Keratin 10 by Regulating Signal Transducer and Activator of Transcription 3 Activation in Human Keratinocytes.

doi: 10.1016/j.jid.2021.05.033

Figure Lengend Snippet: Figure 5. Nuclear IL-33/p-STAT3 confers IL-31 downregulation of K1 and K10 by reducing RunX1. Axs-infected keratinocytes were treated with IL-31, then the mRNA levels of (a) K1 and K10 and (b) RunX1 were measured; (c) the protein levels of p-STAT3, K1, K10, and RunX1 were evaluated. After transfection with siRNA, keratinocytes were treated with IL-31. The mRNA and protein levels of (d) IL-33 and RunX1 and (e) K1, K10, and FLG were determined. 1 denotes IL-31 (‒); 2, 3, and 4 denote IL-31 (þ); 1 and 2 denote cr siRNA; 3 denotes IL-33 siRNA; and 4 denotes IL-33 siRNA þ RunX1 siRNA. Data shown are the mean SD for three wells and are representative of three independent experiments with similar results. *P < 0.05 and #P < 0.05 versus the relevant control group by two- way ANOVA. Ax, adenovirus vector; AxSTAT3F, adenovirus vector expressing dominant-negative signal transducer and activator of transcription 3; cr, control; h, hour; K, keratin; NE, nuclear extract; p-STAT, phosphorylated signal transducer and activator of transcription; siRNA, small interfering RNA; STAT, signal transducer and activator of transcription; WCE, whole-cell extract.

Article Snippet: List of Primary Antibodies Used in the Study Name of Antibodies Catalog Number Provider Usage (Dilution) p44/42 MAPK (ERK1/2) 9102 Cell Signaling Technology (Danvers, MA) Western blotting (1/1,000) Phosphorylated p44/42 MAPK (ERK1/2) (Thr202/Tyr204) 9101 Cell Signaling Technology (Danvers, MA) Western blotting (1/1,000) STAT3 (124H6) 9139 Cell Signaling Technology (Danvers, MA) Western blotting (1/1,000) Phosphorylated STAT3 (Tyr705) (58E12) 9135 Cell Signaling Technology (Danvers, MA) Western blotting (1/1,000) Anti‒IL-33 (human) PM033 MBL (Tokyo, Japan) Western blotting (1/1,000) Immunofluorescence (1/200) IL-33 (4E9) sc-130625 Santa Cruz Biotechnology (Dallas, TX) Western blotting (1/500) (for immunoprecipitation samples) FLG (M-290) sc-30230 Santa Cruz Biotechnology (Dallas, TX) Western blotting (1/500) FLG (AKH1) sc-66192 Santa Cruz Biotechnology (Dallas, TX) Immunofluorescence (1/100) Cytokeratin 1 (E-12) sc-376224 Santa Cruz Biotechnology (Dallas, TX) Western blotting (1/500) Immunofluorescence (1/100) Keratin 10 Ab-2 MS-611-P1 Thermo Fisher Scientific (Yokohama, Japan) Western blotting (1/500) Immunofluorescence (1/100) RUNX1 (A-2) sc-365644 Santa Cruz Biotechnology (Dallas, TX) Western blotting (1/500) Histone H3 2650 Cell Signaling Technology (Danvers, MA) ChIP assay (1/200) Western blotting (1/5,000) GAPDH (0411) sc-47724 Santa Cruz Biotechnology (Dallas, TX) Western blotting (1/2,000) Anti-b Actin (Ac-15) ab6276 Abcam (Tokyo, Japan) Western blotting (1/10,000) Phosphorylated STAT3 (Tyr705) (D3A7) XP 9145 Cell Signaling Technology (Danvers, MA) ChIP assay (1/200) Immunoprecipitation (1/100) Abbreviations: ChIP, chromatin immunoprecipitation; ERK, extracellular signal‒regulated kinase; STAT, signal transducer and activator of transcription; Thr, Threonine; Tyr, Tyrosine. www.jidonline.org 144.e3

Techniques: Infection, Transfection, Control, Plasmid Preparation, Expressing, Dominant Negative Mutation, Small Interfering RNA

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: IAC enrichment coupled with free-label MS was used to define proteins specifically recruited to ligand-bound α V β 6 in HER2+ breast cancer cell lines. ( A and B ) Volcano plots demonstrating enrichment of proteins identified on LAP (α V β 6 integrin–selective ligand; right) and Coll-I (non-α V β 6 integrin binding ligand; left) matrices in (A) HER2-18 and (B) BT474 cells. Statistical analysis: Fisher’s exact test; quantitative method: weighted spectra; significance level: P < 0.05. Significant proteins (dark gray); nonsignificant proteins (light gray); proteins of interest highlighted in purple. ( C and D ) Visual representation of ClueGO cellular compartment GO analyses of proteins significantly enriched on LAP in comparison with Coll-I in (C) HER2-18 and (D) BT474 cells. Colors represent specific merged GO term groups, node size represents level of significance of each GO term, and clustering and edge length represent functionally grouped networks based on kappa score. Yellow boxes highlight the cytoplasmic vesicle GO term cluster. ( E and F ) Top functional subnetworks of proteins significantly enriched on LAP in comparison with Coll-I in (E) HER2-18 and (F) BT474 cells, identified using the OH-PIN algorithm. Colors represent the primary cellular compartment GO term associated with each protein as identified in (C) and (D), respectively. Yellow boxes [(Ea) and (Fa)] highlight the clusters of proteins related to GO term cytoplasmic vesicle, in the top functional subnetwork isolated from each cell line. [(Eb) and (Ec)] Second and third most significant subnetworks in HER2-18 cells. (Fb) All proteins in the cytoplasmic vesicle GO term within the primary functional subnetwork in α V β 6 integrin/LAP-enriched IACs in BT474 cells. All MS data represent three independent experiments. See also figs. S1 (HER2-18) and S3 (BT474) and data files S1 and S2.

Article Snippet: Primary antibodies against RAB5 (Cell Signaling Technology, rabbit mAb #3547), RAB7 (Cell Signaling Technology, rabbit mAb #9367), GDI2 (Thermo Fisher Scientific, rabbit pAb #pa5-48831), HER2 (Cell Signaling Technology, rabbit mAb #2165), pHER2 Y877 (Abcam, rabbit mAb #2241), pHER2 Y1248 (Abcam, rabbit mAb #2247), pHER2 Y1222 (Abcam, rabbit mAb #2243), pHER2 Y1196 (Abcam, rabbit mAb #6942), pHER2 Y1112 (Millipore, mouse mAb #04-294), αV-integrin (Abcam, rabbit mAb #ab179475), β6-integrin (Santa Cruz Biotechnology, goat pAb #sc-6632), β1-integrin (Abcam, rabbit mAb #ab52971), vinculin (Abcam, mouse mAb #ab11194), paxillin (BD, mouse mAb #610051), phospho-Erk 1/2 (p44/42 MAPK) T202/Y204 (pERK1/2) (Cell Signaling Technology, rabbit mAb #137F5), Akt (Cell Signaling Technology, rabbit mAb #4691), pAkt (Cell Signaling Technology, rabbit mAb #4060), GAPDH (Abcam, mouse mAb #ab9484), or β-actin (Sigma-Aldrich, mouse mAb #A3853) were incubated at 4°C overnight.

Techniques: Binding Assay, Comparison, Functional Assay, Isolation

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A ) Immunoblot analysis of β 6 integrin, α V integrin, HER2, paxillin, vinculin, ERK1/2, and GAPDH protein levels in IACs isolated from BT474 cells on LAP, FN, and Coll-I ( N = 3). ( B ) HER2 (magenta) and β 6 integrin (green) immunofluorescence in BT474 cells. Two Z planes of the same cell: (Ba) cell-matrix interface and (Bb) middle Z plane. Dashed boxes: insets. Arrows: membrane-proximal vesicular HER2/β 6 colocalization; scale bars, 10 μm. ( C ) Immunoblot analysis of integrin β 6 , α V , and β 1 and actin (loading control) expression in BT474 cells treated with trastuzumab (10 μg/ml) for 0, 1, and 24 hours ( N = 3). One-way ANOVA, Šídák’s multiple comparison test. ( D ) Immunoblot analysis of total β 6 integrin and HER2 expression, normalized to actin, in trastuzumab-sensitive (Sen) and trastuzumab-resistant (Res) BT474 cells ( N = 3). Two-sided t test, Welch’s correction. ( E ) Flow cytometry analysis of cell surface α V β 6 integrin expression in trastuzumab-sensitive and trastuzumab-resistant BT474 cells [mean fluorescence intensity (MFI) normalized to Sen cells, N = 4]. Two-sided t test. ( F ) Fluorescence analysis of HER2 expression at the plasma membrane of trastuzumab-sensitive and trastuzumab-resistant nonpermeabilized BT474 cells surface labeled with FITC-conjugated HER2 affibody ( N = 3; 44 to 52 cells per condition); scale bar, 10 μm. Two-sided Mann-Whitney test. ( G ) Flow cytometry analysis of HER2 cell surface expression in trastuzumab-sensitive (Sen) and trastuzumab-resistant (Res) BT474 cells (MFI normalized to Sen cells, N = 3). Two-sided t test. ( H and I ) Cell surface expression of α V β 6 integrin (H) and HER2 (I) by flow cytometry in HER2+ breast cancer cells that are endogenously sensitive (white) or resistant (black) to trastuzumab ( N = 4). One-way ANOVA, Dunnett’s multiple comparison test. [(C) to (I)] Data shown are arbitrary units (AU) normalized to control means (untreated trastuzumab-sensitive BT474 cells) ± SEM. Statistical significance: * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: Primary antibodies against RAB5 (Cell Signaling Technology, rabbit mAb #3547), RAB7 (Cell Signaling Technology, rabbit mAb #9367), GDI2 (Thermo Fisher Scientific, rabbit pAb #pa5-48831), HER2 (Cell Signaling Technology, rabbit mAb #2165), pHER2 Y877 (Abcam, rabbit mAb #2241), pHER2 Y1248 (Abcam, rabbit mAb #2247), pHER2 Y1222 (Abcam, rabbit mAb #2243), pHER2 Y1196 (Abcam, rabbit mAb #6942), pHER2 Y1112 (Millipore, mouse mAb #04-294), αV-integrin (Abcam, rabbit mAb #ab179475), β6-integrin (Santa Cruz Biotechnology, goat pAb #sc-6632), β1-integrin (Abcam, rabbit mAb #ab52971), vinculin (Abcam, mouse mAb #ab11194), paxillin (BD, mouse mAb #610051), phospho-Erk 1/2 (p44/42 MAPK) T202/Y204 (pERK1/2) (Cell Signaling Technology, rabbit mAb #137F5), Akt (Cell Signaling Technology, rabbit mAb #4691), pAkt (Cell Signaling Technology, rabbit mAb #4060), GAPDH (Abcam, mouse mAb #ab9484), or β-actin (Sigma-Aldrich, mouse mAb #A3853) were incubated at 4°C overnight.

Techniques: Western Blot, Isolation, Immunofluorescence, Membrane, Control, Expressing, Comparison, Flow Cytometry, Fluorescence, Clinical Proteomics, Labeling, MANN-WHITNEY

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A and B ) Affibody-chase experiments. Cells surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP) to stimulate α V β 6 integrin and trigger α V β 6 endocytosis, or vehicle (Control), 0- to 60-min time course. Quantitation represents cytoplasmic HER2 fluorescence intensity analysis in (A) trastuzumab-sensitive or (B) trastuzumab-resistant BT474 cells ( N = 3; 27 to 50 cells per condition), normalized to control trastuzumab-sensitive BT474 cells (0 min); scale bar, 10 μm. Two-way ANOVA with Šídák’s multiple comparison test. Image intensity increased in (B), relative to (A), due to low cell surface HER2 levels in trastuzumab-resistant cells to highlight internalization differences. ( C ) HER2 (green) and RAB5 (magenta) immunofluorescence in trastuzumab-sensitive and trastuzumab-resistant BT474 cells, treated with soluble LAP, 0 to 60 min ( N = 3; 16 to 28 cells per condition); scale bar, 10 μm. ( Ca ) HER2/RAB5 colocalization quantitation (Pearson’s coefficient ± SEM). Two-way ANOVA with Dunnett’s multiple comparison test. ( D ) Active RAB5 pull-down assays. 0- to 60-min LAP stimulation time course. Quantitation of mean RAB5 activity (pull-down eluate), relative to total RAB5 (lysate) ± SEM ( N = 3), normalized to 0-min trastuzumab-sensitive cells. One-way ANOVA with Dunnett’s multiple comparison test. ( E and F ) Affibody-chase experiments in (E) siControl Trastuzumab-Sensitive or (F) Trastuzumab-Resistant BT474 cells expressing constitutively active RAB5 (RAB5CA), dominant-negative RAB5 (RAB5DN), dominant-negative RAB7 (RAB7DN), or mCherry vector control. Cells were surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP), or vehicle control (control), for 0 or 30 min. Quantitation represents cytoplasmic HER2 fluorescence intensity ( N = 3; 81 to 87 cells per condition); scale bar, 10 μm. One-way ANOVA with Tukey’s multiple comparison test. Representative images in fig. S10 (A and B). Further HER2 internalization analyses: Supplementary Results and fig. S11 (A to D). [(A), (B), and (D) to (F)] Data are arbitrary units (AU) normalized to control means ± SEM. [(A) to (F)] Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Primary antibodies against RAB5 (Cell Signaling Technology, rabbit mAb #3547), RAB7 (Cell Signaling Technology, rabbit mAb #9367), GDI2 (Thermo Fisher Scientific, rabbit pAb #pa5-48831), HER2 (Cell Signaling Technology, rabbit mAb #2165), pHER2 Y877 (Abcam, rabbit mAb #2241), pHER2 Y1248 (Abcam, rabbit mAb #2247), pHER2 Y1222 (Abcam, rabbit mAb #2243), pHER2 Y1196 (Abcam, rabbit mAb #6942), pHER2 Y1112 (Millipore, mouse mAb #04-294), αV-integrin (Abcam, rabbit mAb #ab179475), β6-integrin (Santa Cruz Biotechnology, goat pAb #sc-6632), β1-integrin (Abcam, rabbit mAb #ab52971), vinculin (Abcam, mouse mAb #ab11194), paxillin (BD, mouse mAb #610051), phospho-Erk 1/2 (p44/42 MAPK) T202/Y204 (pERK1/2) (Cell Signaling Technology, rabbit mAb #137F5), Akt (Cell Signaling Technology, rabbit mAb #4691), pAkt (Cell Signaling Technology, rabbit mAb #4060), GAPDH (Abcam, mouse mAb #ab9484), or β-actin (Sigma-Aldrich, mouse mAb #A3853) were incubated at 4°C overnight.

Techniques: Labeling, Control, Quantitation Assay, Fluorescence, Comparison, Immunofluorescence, Activity Assay, Expressing, Dominant Negative Mutation, Plasmid Preparation

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A and B ) Affibody-chase experiments: siControl-transfected or siGDI2-transfected BT474 cells surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP) to stimulate α V β 6 integrin and trigger α V β 6 endocytosis, or vehicle (Control), 0- to 60-min time course. Quantitation represents cytoplasmic HER2 fluorescence intensity analysis in (A) trastuzumab-sensitive or (B) trastuzumab-resistant BT474 cells ( N = 3; 74 to 160 cells per condition); scale bars, 10 μm. Two-way ANOVA with Tukey’s multiple comparison test. Image intensity increased in (B), relative to (A), due to low cell surface HER2 levels in trastuzumab-resistant cells to highlight internalization differences. ( C ) GDI2 (green) and RAB5 (magenta) immunofluorescence in trastuzumab-sensitive and trastuzumab-resistant BT474 cells ( N = 3; >120 cells per condition); scale bars, 5 μm. GDI2/RAB5 colocalization quantitation (Pearson’s coefficient ± SEM), two-sided t test. ( D ) Role of GDI2 in α V β 6 -dependent RAB5 activity modulation. Trastuzumab-sensitive and trastuzumab-resistant BT474 cells transfected with siRNA against GDI2 (siGDI2 #1 and #2) or control siRNA. 0- to 60-min LAP stimulation time course. Quantitation of mean RAB5 activity (pull-down eluate), relative to total RAB5 (lysate) ± SEM ( N = 3), normalized to 0-min trastuzumab-sensitive cells. N = 4 independent replicate experiments. Two-way ANOVA with Šídák’s multiple comparison tests. ( E ) Haptotactic migration analysis of BT474 cells (Trastuzumab-Sensitive and Trastuzumab-Resistant) in Transwell coated with FN or BSA as a negative control. Cells were transfected with siRNA against GDI2 (siGDI2 #1 and #2) or siRNA control. Migration was assessed over 24 hours in the presence or absence of α V β 6 integrin blocking antibody or trastuzumab. Data shown are means ± SEM ( N = 3). One-way ANOVA with Šídák’s multiple comparison tests. [(A), (B), (D), and (E)] Data are arbitrary units (AU) normalized to control means ± SEM. [(A) to (E)] Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Primary antibodies against RAB5 (Cell Signaling Technology, rabbit mAb #3547), RAB7 (Cell Signaling Technology, rabbit mAb #9367), GDI2 (Thermo Fisher Scientific, rabbit pAb #pa5-48831), HER2 (Cell Signaling Technology, rabbit mAb #2165), pHER2 Y877 (Abcam, rabbit mAb #2241), pHER2 Y1248 (Abcam, rabbit mAb #2247), pHER2 Y1222 (Abcam, rabbit mAb #2243), pHER2 Y1196 (Abcam, rabbit mAb #6942), pHER2 Y1112 (Millipore, mouse mAb #04-294), αV-integrin (Abcam, rabbit mAb #ab179475), β6-integrin (Santa Cruz Biotechnology, goat pAb #sc-6632), β1-integrin (Abcam, rabbit mAb #ab52971), vinculin (Abcam, mouse mAb #ab11194), paxillin (BD, mouse mAb #610051), phospho-Erk 1/2 (p44/42 MAPK) T202/Y204 (pERK1/2) (Cell Signaling Technology, rabbit mAb #137F5), Akt (Cell Signaling Technology, rabbit mAb #4691), pAkt (Cell Signaling Technology, rabbit mAb #4060), GAPDH (Abcam, mouse mAb #ab9484), or β-actin (Sigma-Aldrich, mouse mAb #A3853) were incubated at 4°C overnight.

Techniques: Transfection, Labeling, Control, Quantitation Assay, Fluorescence, Comparison, Immunofluorescence, Activity Assay, Migration, Negative Control, Blocking Assay

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A ) Trastuzumab-Sensitive Cells: GDI2 is recruited to sites proximal to α V β 6 IACs and coordinates HER2 and α V β 6 trafficking and signaling by locally modulating RAB5 activity. GDI2-mediated cross-talk between α V β 6 and HER2 affects membrane availability of both receptors, ultimately influencing migration, invasion, and TGFβ activation. ( B ) Trastuzumab-Resistant Cells: GDI2 is excluded from α V β 6 IACs, leading to dysregulation of RAB5 activation dynamics, followed by increased RAB7 activation. Consequently, HER2/α V β 6 cross-talk is impaired, altering receptor trafficking dynamics and disrupting bioavailability of both HER2 and α V β 6 integrin at the plasma membrane. This dysregulation further affects TGFβ activation, resulting in increased cell invasiveness and metastatic potential. Overall, these changes may increase the ability of cells to evade HER2 targeting drugs.

Article Snippet: Primary antibodies against RAB5 (Cell Signaling Technology, rabbit mAb #3547), RAB7 (Cell Signaling Technology, rabbit mAb #9367), GDI2 (Thermo Fisher Scientific, rabbit pAb #pa5-48831), HER2 (Cell Signaling Technology, rabbit mAb #2165), pHER2 Y877 (Abcam, rabbit mAb #2241), pHER2 Y1248 (Abcam, rabbit mAb #2247), pHER2 Y1222 (Abcam, rabbit mAb #2243), pHER2 Y1196 (Abcam, rabbit mAb #6942), pHER2 Y1112 (Millipore, mouse mAb #04-294), αV-integrin (Abcam, rabbit mAb #ab179475), β6-integrin (Santa Cruz Biotechnology, goat pAb #sc-6632), β1-integrin (Abcam, rabbit mAb #ab52971), vinculin (Abcam, mouse mAb #ab11194), paxillin (BD, mouse mAb #610051), phospho-Erk 1/2 (p44/42 MAPK) T202/Y204 (pERK1/2) (Cell Signaling Technology, rabbit mAb #137F5), Akt (Cell Signaling Technology, rabbit mAb #4691), pAkt (Cell Signaling Technology, rabbit mAb #4060), GAPDH (Abcam, mouse mAb #ab9484), or β-actin (Sigma-Aldrich, mouse mAb #A3853) were incubated at 4°C overnight.

Techniques: Activity Assay, Membrane, Migration, Activation Assay, Clinical Proteomics

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A ) Differential gene expression data (RNA-seq) for the GDI2 / RAB5A / RAB7A / ERBB2 / ITGB6 cluster in normal breast tissue ( n = 403; light gray) and breast invasive carcinoma ( n = 1097; dark gray). Data were extracted from the TNMplot database ( tnmplot.com ). Black lines in violin blots represent the median. Mann-Whitney test. ( B ) Volcano plot showing statistical analysis (ANOVA) of RNA-seq gene expression data of patients with HER2+ breast cancer from the METABRIC cohort expressing high (Right) and low (Left) levels of ITGB6 (Q1 versus Q4). Significant genes (dark gray); nonsignificant genes (light gray); relevant genes are highlighted in purple. ( C ) Visual representation of GO terms analysis (ClueGO, cellular compartment) of genes highly and significantly expressed in tumors expressing high levels of ITGB6 (Q4). Colors represent specific merged GO term groups, node size represents the level of significance of each GO term, and clustering and edge length represent functionally grouped networks based on kappa score. ( D ) OS of patients with HER2+ breast cancer and with high (above median) expression of ITGB6 , expressing high (red) or low (black) levels of GDI2 , ERBB2 , RAB5A , and RAB7A . ( E and F ) Differential ITGB6 gene expression (gene chip) in patients with HER2+ breast cancer subdivided according to therapeutic response to trastuzumab. (E) Initial pathological complete response (responder) versus residual disease after completing therapy (nonresponder) ( n = 77 patients). (F) RFS at 5 years (responder) versus samples relapsed before 5 years (nonresponder) ( n = 24 patients). Two-sided Student’s t test. [(A), (E), and (F)] Statistical significance: * P < 0.05; **** P < 0.0001.

Article Snippet: Primary antibodies against RAB5 (Cell Signaling Technology, rabbit mAb #3547), RAB7 (Cell Signaling Technology, rabbit mAb #9367), GDI2 (Thermo Fisher Scientific, rabbit pAb #pa5-48831), HER2 (Cell Signaling Technology, rabbit mAb #2165), pHER2 Y877 (Abcam, rabbit mAb #2241), pHER2 Y1248 (Abcam, rabbit mAb #2247), pHER2 Y1222 (Abcam, rabbit mAb #2243), pHER2 Y1196 (Abcam, rabbit mAb #6942), pHER2 Y1112 (Millipore, mouse mAb #04-294), αV-integrin (Abcam, rabbit mAb #ab179475), β6-integrin (Santa Cruz Biotechnology, goat pAb #sc-6632), β1-integrin (Abcam, rabbit mAb #ab52971), vinculin (Abcam, mouse mAb #ab11194), paxillin (BD, mouse mAb #610051), phospho-Erk 1/2 (p44/42 MAPK) T202/Y204 (pERK1/2) (Cell Signaling Technology, rabbit mAb #137F5), Akt (Cell Signaling Technology, rabbit mAb #4691), pAkt (Cell Signaling Technology, rabbit mAb #4060), GAPDH (Abcam, mouse mAb #ab9484), or β-actin (Sigma-Aldrich, mouse mAb #A3853) were incubated at 4°C overnight.

Techniques: Gene Expression, RNA Sequencing, MANN-WHITNEY, Expressing, Clinical Proteomics

Journal: Oncology reports

Article Title: Erb‑B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53‑responsive microRNA‑3184‑5p in cervical cancer cells.

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: Figure 1. ERBB2 expression is upregulated in patient‑derived cervical cancer tissues and is associated with a poor prognosis. (A) RT‑qPCR and (B) WB analysis of ERBB2 transcript and protein expression, respectively, in patient‑derived cervical cancer tissues (n=65) vs. matched healthy cervical tissues (n=65). Data were analyzed via Wilcoxon signed‑rank test. (C) RT‑qPCR and (D) WB analysis of ERBB2 transcript and protein expression, respectively, in stage I/II vs. stage III/IV patient‑derived cervical cancer tissues (n=43 stage I/II; n=22 Stage III/IV). Data were analyzed via Mann‑Whitney U test. (E) RT‑qPCR and (F) WB analysis of ERBB2 transcript and protein expression, respectively, in lymph node metastatic and non‑metastatic patient‑derived cervical cancer biopsies [n=46 lymph node (‑); n=19 lymph node (+)]. Data were analyzed via Mann‑Whitney U test. (G) Survival analysis using the Kaplan‑Meier method according to high (above the median) or low (below the median) ERBB2 mRNA expression (n=32 in each cohort). The P‑value was calculated using the log‑rank test. For purposes of comparison across cohorts, the median ERBB2 mRNA and protein expression levels (normalized to the RT‑qPCR housekeeping control and WB loading control GAPDH) in the normal cohort have been set to 1.0. Data in box plots are expressed as the median ± IQRs (boxes) and absolute ranges (whiskers). n=3. **P<0.01. RT‑qPCR, reverse transcription‑quantitative PCR; WB, western blotting; ERBB2, Erb‑B2 Receptor Tyrosine Kinase 2; Pt, patient.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3'-UTR of human ERBB2 (ERBB2-3'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Expressing, Mann-Whitney U-Test, Comparison, Control, Western Blot

Journal: Oncology reports

Article Title: Erb‑B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53‑responsive microRNA‑3184‑5p in cervical cancer cells.

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: Figure 2. ERBB2 overexpression in cervical cancer cell lines stimulates viability, invasion and sphere‑formation. (A) Confirmation of ERBB2 KD in siERBB2 cells and OE in ERBB2 vec cells via WB. GAPDH was used as the loading control. (B) Invasion of siERBB2 vs. siCtrl cells via Transwell assay. (C) Invasion of ERBB2 vec vs. Ctrl vec cells via Transwell assay. (D) Cellular viability of siERBB2, siCtrl, ERBB2 vec and Ctrl vec cells quantified using a Cell Counting Kit‑8. (E) Sphere‑formation of siERBB2 vs. siCtrl cells. (F) Sphere‑formation of ERBB2 vec vs. Ctrl vec cells. (G) Analysis of metastasis‑associated and cancer stem cell biomarkers mRNA and protein expression in siERBB2, siCtrl, ERBB2 vec and Ctrl vec cells via RT‑qPCR and WB, respectively. GAPDH was used as the RT‑qPCR housekeeping control and WB loading control. Data are expressed as the mean ± SEM (n=3). **P<0.01 vs. siCtrl or Ctrl vec analyzed via unpaired Student's t‑test. KD, knockdown; OE, overexpression; WB, western blotting; RT‑qPCR, reverse transcription‑quantitative PCR; si, small interfering; Ctrl, control; vec, vector; ERBB2, Erb‑B2 Receptor Tyrosine Kinase 2.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3'-UTR of human ERBB2 (ERBB2-3'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Over Expression, Control, Transwell Assay, CCK-8 Assay, Expressing, Knockdown, Western Blot, Plasmid Preparation

Journal: Oncology reports

Article Title: Erb‑B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53‑responsive microRNA‑3184‑5p in cervical cancer cells.

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: Figure 3. ERBB2 controls cervical cancer cell viability and invasion by regulating PIK3CA protein expression. (A) Schematic diagram of the ERBB2‑ERRB3 complex interacting with PI3K(p85), thereby promoting the downstream phosphorylation of AKT and mTOR. (B) IP in cervical cancer cell lysates with antibodies against ERBB3 or IgG control. Expression levels of ERBB3, ERBB2 and PI3K(p85) in the IP fraction were assessed via WB. PIK3CA mRNA expression in transfected (C) HeLa and (D) SiHa cells assessed via RT‑qPCR. GAPDH was used as the housekeeping control. PIK3CA, p‑AKT/AKT and p‑mTOR/mTOR protein expression in transfected (E) HeLa and (F) SiHa cells assessed via WB. GAPDH was used as the loading control. (G) Invasion of transfected HeLa cells assessed via Transwell assay. (H) Cellular viability of transfected HeLa cells quantified using Cell Counting Kit‑8. (I) Sphere‑formation of transfected HeLa cells. Data are expressed as the mean ± SEM (n=3). *P<0.05 and **P<0.01 vs. siCtrl or Ctrl vec; †P<0.05 and ††P<0.01 vs. siERBB2 or ERBB2 vec. Data were analyzed via one‑way ANOVA. IP, immunoprecipitation; WB, western blotting; RT‑qPCR, reverse transcription‑quantitative PCR; si, small interfering; vec, vector; ERBB2, Erb‑B2 Receptor Tyrosine Kinase 2; p‑, phosphorylated; PIK3CA, phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit α.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3'-UTR of human ERBB2 (ERBB2-3'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Expressing, Phospho-proteomics, Control, Transfection, Transwell Assay, CCK-8 Assay, Immunoprecipitation, Western Blot, Plasmid Preparation

Journal: Oncology reports

Article Title: Erb‑B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53‑responsive microRNA‑3184‑5p in cervical cancer cells.

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: Figure 4. miR‑3184‑5p attenuates cervical cancer cell viability and invasion by targeting ERBB2. (A) Putative binding location for miR‑3184‑5p on ERBB2 3'‑UTR via TargetScan analysis. (B) miR‑3184‑5p expression in HeLa and SiHa cervical cancer cell lines compared with in the non‑cancerous human H8 cervical epithelial cell line assessed via RT‑qPCR. U6 was used as the housekeeping control. **P<0.01 vs. H8; ††P<0.01 vs. SiHa. Luciferase reporter assay of ERBB2‑3'‑UTRWT or ERBB2‑3'‑UTRMU in (C) HeLa or (D) SiHa cells transfected with miR‑3184‑5p mimic or inhibitor, respectively. **P<0.01 vs. Ctrl mimic or Ctrl inhib. WB of (E) HeLa and (F) SiHa cells transfected with miR‑3184‑5p mimic or inhibitor, respectively. (G) Invasion of transfected HeLa cells assessed via Transwell assay. (H) Cellular viability of transfected HeLa cells quantified using Cell Counting Kit‑8. (I) Sphere‑formation of transfected HeLa cells. Data are expressed as the mean ± SEM (n=3). **P<0.01 vs. Ctrl vec; ††P<0.01 vs. miR‑3184‑5p mimic. Data were analyzed via one‑way ANOVA. UTR, untranslated region; WT, wild‑type; MU, mutant; Ctrl, control; inhib, inhibitor; WB, western blotting; RT‑qPCR, reverse transcription‑quantitative PCR; miR, microRNA; vec, vector; ERBB2, Erb‑B2 Receptor Tyrosine Kinase 2; PIK3CA, phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit α.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3'-UTR of human ERBB2 (ERBB2-3'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Binding Assay, Expressing, Control, Luciferase, Reporter Assay, Transfection, Inhibition, Transwell Assay, CCK-8 Assay, Mutagenesis, Western Blot, Plasmid Preparation

Journal: Oncology reports

Article Title: Erb‑B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53‑responsive microRNA‑3184‑5p in cervical cancer cells.

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: Figure 5. p53‑activating Mithramycin A boosts miR‑3184‑5p expression, which lowers ERBB2 expression and attenuates viability and invasion of cervical cancer cell lines. (A) p53, p21 and ERBB2 protein expression in cervical cancer cultures incubated with MM or vehicle (DMSO) assessed via WB. GAPDH was used as the loading control. (B) miR‑3184‑5p expression in cervical cancer cultures incubated with MM or vehicle assessed via RT‑qPCR. U6 was used as the housekeeping control. (C) p53 and ERBB2 protein expression in cervical cancer cultures transfected with a p53 overexpression plasmid or empty plasmid control assessed via WB. GAPDH was used as the loading control. (D) miR‑3184‑5p expression in cervical cancer cultures transfected with a p53 overexpression plasmid or empty plasmid control assessed via RT‑qPCR. U6 was used as the housekeeping control. (E) Representative images of Transwell and sphere‑formation assays in (E) HeLa and (F) SiHa cells, and quantitative analysis of viability, invasion and sphere‑formation of cells treated with MM or vehicle. (G) Schematic diagram of the p53 activator MM rescuing miR‑3184‑5p expression, thereby suppressing ERBB2 transcription. This attenuates PIK3CA activity, which stimulates cervical cancer cell viability, invasion and sphere‑formation. Data are expressed as the mean ± SEM (n=3). **P<0.01 analyzed via unpaired Student's t‑test. MM, Mithramycin A; Ctrl, control; WB, western blotting; RT‑qPCR, reverse transcription‑quantitative PCR; miR, microRNA; vec, vector; ERBB2, Erb‑B2 Receptor Tyrosine Kinase 2; p‑, phosphorylated; PIK3CA, phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit α.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3'-UTR of human ERBB2 (ERBB2-3'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Expressing, Incubation, Control, Transfection, Over Expression, Plasmid Preparation, Activity Assay, Western Blot

Journal: British Journal of Cancer

Article Title: Loss of FOXO1 promotes gastric tumour growth and metastasis through upregulation of human epidermal growth factor receptor 2/neu expression

doi: 10.1038/bjc.2015.273

Figure Lengend Snippet: The in vitro effect of FOXO1 on EMT, cell migration and invasion of SNU-638 cells, MKN45 cells and SNU-216 cells. ( A and E ) The expressions of E-cadherin and Snail in GC cells expressing either control shRNA (shCtrl) or FOXO1 shRNA (shFOXO1) were determined by immunoblot analysis. ( B ) Changes in the organisation of the actin cytoskeleton. Cells were stained with Alexa Fluor 633-conjugated phalloidin to visualise F-actin (red), and the cell nuclei were visualised by DAPI staining (blue). Arrows indicate the FITC-labelled filopodia-like projections. Photographs were taken with a fluorescence microscope ( × 400 magnification). ( C ) Immunofluoroscence stainings for FOXO1 (red), E-cadherin (green) and Snail (red) were performed. Cell nuclei were visualised by DAPI staining (blue) ( × 400 magnification). ( D and F ) The effect of FOXO1 silencing on cell migration/invasion was evaluated by Transwell migration assay and cell invasion assay followed by cell viability assessment using the crystal violet assay. Representative images of migrated/invasive cells taken 48 h after plating into a Transwell insert are on the left, and the quantification of migrated/invasive cells is on the right. The motility/invasiveness of cells expressing shCtrl corresponded to 1. Bars represent mean±s.d. ( n =4). * P <0.05, compared with shCtrl. ( G and H ) SNU-216 cells were transfected with either control plasmid pcDNA3 or pFOXO1A3 (FOXO1A3). ( G ) The effect of FOXO1A3 transfection into GC cells on the expression of E-cadherin and Snail was determined by immunoblot analysis. ( H ) The effect of FOXO1A3 expression in GC cells on the cell migration and invasion was determined as described above. Bars represent mean±s.d. ( n =4). * P <0.05, compared with vector control (pcDNA3).

Article Snippet: Control plasmid pcDNA3 and expression plasmids containing either human FOXO1A3 mutant gene (Addgene plasmid 13508) or HER2 wild-type (WT) gene (Addgene plasmid 16257) were purchased from Addgene Incorp.

Techniques: In Vitro, Migration, Expressing, Control, shRNA, Western Blot, Staining, Fluorescence, Microscopy, Transwell Migration Assay, Invasion Assay, Crystal Violet Assay, Transfection, Plasmid Preparation

Journal: British Journal of Cancer

Article Title: Loss of FOXO1 promotes gastric tumour growth and metastasis through upregulation of human epidermal growth factor receptor 2/neu expression

doi: 10.1038/bjc.2015.273

Figure Lengend Snippet: Effects of FOXO1 silencing on HER2 expression in GC cells and xenograft tumours. ( A ) FOXO1 was downregulated by transfection of FOXO1 shRNA (shFOXO1) into SNU-638 and MKN45 cells. Immunoblot analysis (WB) and RT–PCR showed that FOXO1 shRNA transfection increased the expressions of HER2 protein and mRNA compared with control shRNA (shCtrl) transfection. ( B ) FOXO1 expression in SNU-216 cells was upregulated by transfection of FOXO1A3. WB and RT–PCR showed that FOXO1A3 transfection decreased HER2 protein and mRNA expressions compared with pcDNA3 transfection. ( C ) ChIP for the binding of FOXO1 to the HER2 promoter in SNU-638 cells. Chromatin was cross-linked and immunoprecipitated using anti-FOXO1. Precipitated DNAs were amplified and quantified by real-time PCR using two primers (PM1 and PM2). The results are expressed as percentages of the input level. Bars represent mean±s.d. ( n =3). * P <0.05, compared with shCtrl. ( D ) Representative features of immunohistochemical staining for FOXO1 (upper panels) and HER2 (lower panels). Sections obtained from orthotopic xenograft tumours showed that shFOXO1-expressing tumours had higher HER2 expression than shCtrl-expressing tumours ( × 400 magnification).

Article Snippet: Control plasmid pcDNA3 and expression plasmids containing either human FOXO1A3 mutant gene (Addgene plasmid 13508) or HER2 wild-type (WT) gene (Addgene plasmid 16257) were purchased from Addgene Incorp.

Techniques: Expressing, Transfection, shRNA, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Binding Assay, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: JWA loss promotes cell migration and cytoskeletal rearrangement by affecting HER2 expression and identifies a high-risk subgroup of HER2-positive gastric carcinoma patients

doi: 10.18632/oncotarget.9211

Figure Lengend Snippet: A. The mRNA levels of HER2, JWA and a panel of putative transcription factors that regulate HER2 were determined by qPCR after HGC-27 cells were transfected with si-JWA and scramble control RNA. B, C. The mRNA and protein levels of HER2, JWA and PEA3 were identified by qPCR or western blot analyses in NCI-N87 cells transfected with FLAG-JWA or vector as well as in HGC-27 cells transfected with si-JWA or scramble control. * P<0.05; ** P<0.01; Student's t-test. D. PEA3 levels in nuclear and cytoplasmic extracts were confirmed by western blotting in JWA-overexpressing NCI-N87 cells and JWA-silenced HGC-27 cells. Actin and Histone H3 were used as cytoplasmic and nuclear loading controls, respectively. E. Four and six micrograms of nuclear protein were extracted from JWA-knockdown HGC-27 cells and JWA-overexpressing NCI-N87 cells to perform EMSA with a biotinylated oligonucleotide containing the PEA3-binding site and its competitive probe. F. NCI-N87 cells were transiently co-transfected with 2.5 μg (upper panel) or different amounts of FLAG-JWA (lower panel) and 2.5 μg of HER2 luciferase reporter promoter plasmids without (pNeuLite) or with the PEA3-binding site mutation (PEA3mut). The cells were lysed 36 h after transfection, and the luciferase activity was measured. The relative HER2 promoter activity was calculated relative to the activity of the wild-type promoter in vector cells (defined as 100%) after normalization to pRL-CMV. * P<0.05 and ** P<0.01 compared with vector pNeuLite activity.

Article Snippet: The pNeuLite plasmids contain the core promoter of the HER2 gene, and the PEA3 mt plasmids have a mutated PEA3-binding motif in the HER2 promoter (kindly provided by Prof. Mien-Chie Hung; Addgene, MA, USA).

Techniques: Transfection, Control, Western Blot, Plasmid Preparation, Knockdown, Binding Assay, Luciferase, Mutagenesis, Activity Assay

Journal: Oncotarget

Article Title: JWA loss promotes cell migration and cytoskeletal rearrangement by affecting HER2 expression and identifies a high-risk subgroup of HER2-positive gastric carcinoma patients

doi: 10.18632/oncotarget.9211

Figure Lengend Snippet: A, B. NCI-N87 cells were transfected with FLAG-JWA or vector for 36 h and then treated with U0126 (25 μg/ml) or DMSO for 6 h and EGF (100 ng/ml) for 20 min. HER2 luciferase reporter promoter plasmids (pNeuLite) were co-transfected with vector or FLAG-JWA into NCI-N87 cells. The luciferase activity was normalized to pRL-CMV. The HER2 promoter luciferase activity in vector-treated cells was used as a control to calculate the relative HER2 luciferase activity (A). The PEA3, p-ERK, JWA, and HER2 protein levels were examined by western blot analysis (B). C. The MEK (U0126; 25 μM) and/or PI3K (LY294002; 50 μM) inhibitors or control DMSO were added to the cells for 6 h, and the cells were then transfected with FLAG-JWA or vector for 36 h. The cells were then harvested to detect HER2, p-AKT, p-ERK, p-PAK1, FAK, COX2, or FLAG by immunoblotting. The data in the bar graphs represent the mean and SD of three independent experiments. * P<0.05 and ** P<0.01; Student's t-test. Arrows indicate the interested band.

Article Snippet: The pNeuLite plasmids contain the core promoter of the HER2 gene, and the PEA3 mt plasmids have a mutated PEA3-binding motif in the HER2 promoter (kindly provided by Prof. Mien-Chie Hung; Addgene, MA, USA).

Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay, Control, Western Blot