Journal: The Journal of Cell Biology

Article Title: Active and inactive β1 integrins segregate into distinct nanoclusters in focal adhesions

doi: 10.1083/jcb.201707075

Figure Lengend Snippet: Anti–β1 integrin mAbs

Article Snippet: Primary abs used for immunostaining were rat anti–β1 integrin mAb 9EG7 (BD; ; ; ), mAb 13 (BD; ; ; ), and mAb AIIB2 (Developmental Studies Hybridoma Bank; ; ; ); mouse anti–β1 integrin mAb K20 (Santa Cruz Biotechnology, Inc.; ; ), mAb Huts-4 (Chemicon; ; ), and mAb 12G20 (Abcam; ; ); mouse antitalin mAb 8d4 (Sigma-Aldrich); and rabbit polyclonal abs antivinculin (V4139; Sigma-Aldrich) and anti–kindlin-2 (ab74030; Abcam).

Techniques:

Journal: The Journal of Cell Biology

Article Title: Active and inactive β1 integrins segregate into distinct nanoclusters in focal adhesions

doi: 10.1083/jcb.201707075

Figure Lengend Snippet: Nanoorganization of β1 integrins within FAs. (A) Representative images of an Hs578T cell labeled by anti–total β1 integrin mAb K20 (OG488) acquired by confocal microscopy (left) and STED (middle). The white box in the middle panel is zoomed in the right inset. (B) Representative intensity profiles along an individual adhesion (green/red lines in A; n = 10, one profile per image) reveal distinct intensity peaks by STED (red) but not by confocal microscopy (green). (C) Spatial distribution of identified intensity peaks within adhesions in the STED image from A. (D) Representative image of an Hs578T cell labeled with anti–β1 integrin mAb K20 (Alexa Fluor 405 through Alexa Fluor 647) acquired by TIRF (left) and STORM (middle). The white box is zoomed in on the right and shows clusters identified by DBSCAN. Bars: (main images) 5 µm; (insets) 500 nm.

Article Snippet: Primary abs used for immunostaining were rat anti–β1 integrin mAb 9EG7 (BD; ; ; ), mAb 13 (BD; ; ; ), and mAb AIIB2 (Developmental Studies Hybridoma Bank; ; ; ); mouse anti–β1 integrin mAb K20 (Santa Cruz Biotechnology, Inc.; ; ), mAb Huts-4 (Chemicon; ; ), and mAb 12G20 (Abcam; ; ); mouse antitalin mAb 8d4 (Sigma-Aldrich); and rabbit polyclonal abs antivinculin (V4139; Sigma-Aldrich) and anti–kindlin-2 (ab74030; Abcam).

Techniques: Labeling, Confocal Microscopy

Journal: The Journal of Cell Biology

Article Title: Active and inactive β1 integrins segregate into distinct nanoclusters in focal adhesions

doi: 10.1083/jcb.201707075

Figure Lengend Snippet: Distinct organization of active and inactive β1 integrin nanoclusters within FAs. (A) Left: Representative STED image of an Hs578T cell labeled with antiactive β1 integrin mAb 9EG7 (rhodamine). Right: Spatial distribution of detected intensity peaks within FAs. (B) Left: Representative STED image of Hs578T cells labeled with antiinactive β1 integrin mAb AIIB2 (rhodamine). Right: Spatial distribution of detected intensity peaks within FAs. (C) Bars show FAs scored for linear versus unstructured patterns in labelings of mAbs K20 ( n = 23 images), 9EG7 ( n = 21 images), and AIIB2 ( n = 18 images), displayed as mean percentages ± SEM. (D) Automated linearity analysis. Left: Means ± SD of fitted RANSAC lines needed to deplete all segmented clusters per FA for K20 ( n = 13 cells), 9EG7 ( n = 11 cells), and AIIB2 ( n = 11 cells) labelings. Right: Mean ± SD number of clusters per fitted RANSAC line for the same experimental set. Kolmogorov-Smirnov test: *, P < 0.05; **, P < 0.01. (E) Bars show median NND (quartile distribution in boxes; decile distributions in whiskers) of STED-identified clusters for mAb 9EG7 ( n = 19 images) and mAb AIIB2 ( n = 19) labelings within FAs. The two distributions display different variances. (F and G) Left: Representative STORM image of Hs578T cells labeled with mAb 9EG7 (F) or mAb AIIB2 (G; Alexa Fluor 405 through Alexa Fluor 647). White boxes indicate zooms shown on right. Red circles show DBSCAN-identified clusters in zoomed areas. Bars: (A, B, F, and G,main images) 5 µm; (F and G, insets) 500 nm. (H) Error bars show median NNDs (quartile distribution in boxes; decile distribution in whiskers) of β1 integrin clusters in STORM images of mAb 9EG7 ( n = 12 images) and mAb AIIB2 ( n = 14 images) labeling. The two distributions display different variances. (I and J) The bars show median cluster size (I) and median number of cluster localizations (J) of STORM images (quartile distribution in boxes; decile distribution in whiskers) of mAb 9EG7 ( n = 14 images) and mAb AIIB2 ( n = 14) labeling within FAs. t test: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Article Snippet: Primary abs used for immunostaining were rat anti–β1 integrin mAb 9EG7 (BD; ; ; ), mAb 13 (BD; ; ; ), and mAb AIIB2 (Developmental Studies Hybridoma Bank; ; ; ); mouse anti–β1 integrin mAb K20 (Santa Cruz Biotechnology, Inc.; ; ), mAb Huts-4 (Chemicon; ; ), and mAb 12G20 (Abcam; ; ); mouse antitalin mAb 8d4 (Sigma-Aldrich); and rabbit polyclonal abs antivinculin (V4139; Sigma-Aldrich) and anti–kindlin-2 (ab74030; Abcam).

Techniques: Labeling

Journal: The Journal of Cell Biology

Article Title: Active and inactive β1 integrins segregate into distinct nanoclusters in focal adhesions

doi: 10.1083/jcb.201707075

Figure Lengend Snippet: Distinct localization of active and inactive β1 integrins within FAs. (A) Representative confocal, STED, and STORM images of Hs578T cells colabeled with antiactive β1 integrin mAb 12G10 (OG488 or Cy3; Alexa Fluor 647) and antiinactive β1 integrin mAb AIIB2 (rhodamine or Alexa Fluor 405 through Alexa Fluor 647). White boxes indicate zoomed areas on the right. Bars: (main images) 5 µm; (insets) 2 µm. (B–D) Bars show mean Pearson’s correlation coefficients (± SD) obtained by confocal (B), STED (C), and STORM (D) images colabeled with mAb 12G10 and mAb AIIB2. Confocal, n = 10 images; STED, n = 20; STORM, n = 6. mAb 12G10 labelings targeted by two differently colored secondary abs were used as control for maximal colocalization. Confocal, n = 9 images; STED, n = 13; STORM, n = 6. (E) The three left bars show Hs578T cells labeled by mAbs AIIB2 ( n = 18 images), K20 ( n = 11), or 12G10 ( n = 13) targeted with two differently colored secondary abs each as controls for maximal colocalization. The remaining bars show mean Pearson’s correlation coefficients (± SD) of STED images of Hs578T cells colabeled by mAb 12G10 and mAb 9EG7 ( n = 12 images); mAb Huts-4 and mAb 9EG7 ( n = 11); mAb K20 and mAb 9EG7 ( n = 14); mAb K20 and mAb AIIB2 ( n = 17); mAb 12G10 and mAb AIIB2 ( n = 20); mAb 12G10 and mAb 13 ( n = 11); mAb Huts-4 and mAb AIIB2 ( n = 11); and mAb Huts-4 and mAb 13 ( n = 5). The individual colocalizations were statistically compared with the control with the lowest coefficient (12G10), providing the most stringent comparison. The mAb AIIB2 versus mAb12G10 combination was duplicated from C to facilitate direct comparison. (F) Bars show mean percentages ± SD of overlapping nanoclusters segmented in STED images of Hs578T cells colabeled with mAb 12G10 and mAb AIIB2 ( n = 20 images). Hs578T cells labeled using mAb K20 and two differently colored secondary abs both targeting mAb K20 were used as control for maximal overlap. n = 9 images. t test: *, P ≤ 0.05; ***, P ≤ 0.001.

Article Snippet: Primary abs used for immunostaining were rat anti–β1 integrin mAb 9EG7 (BD; ; ; ), mAb 13 (BD; ; ; ), and mAb AIIB2 (Developmental Studies Hybridoma Bank; ; ; ); mouse anti–β1 integrin mAb K20 (Santa Cruz Biotechnology, Inc.; ; ), mAb Huts-4 (Chemicon; ; ), and mAb 12G20 (Abcam; ; ); mouse antitalin mAb 8d4 (Sigma-Aldrich); and rabbit polyclonal abs antivinculin (V4139; Sigma-Aldrich) and anti–kindlin-2 (ab74030; Abcam).

Techniques: Labeling

Journal: The Journal of Cell Biology

Article Title: Active and inactive β1 integrins segregate into distinct nanoclusters in focal adhesions

doi: 10.1083/jcb.201707075

Figure Lengend Snippet: Colocalization of talin, kindlin-2, and vinculin with active and inactive β1 integrins within FAs. (A) Left: Representative STED images of Hs578T cells labeled with antiactive β1 integrin mAb 9EG7 (left) or antiinactive β1 integrin mAb AIIB2 (right; both rhodamine) and antitalin mAb, anti–kindlin-2 polyclonal ab, or antivinculin polyclonal ab (all OG488). White boxes indicate zoomed areas. Bars: (main images) 5 µm; (insets) 2 µm. (B) Bars show mean Pearson’s correlation coefficients (± SD) of STED images of Hs578T cells colabeled as indicated. n = 10–11 images for each combination. t test: ***, P ≤ 0.001.

Article Snippet: Primary abs used for immunostaining were rat anti–β1 integrin mAb 9EG7 (BD; ; ; ), mAb 13 (BD; ; ; ), and mAb AIIB2 (Developmental Studies Hybridoma Bank; ; ; ); mouse anti–β1 integrin mAb K20 (Santa Cruz Biotechnology, Inc.; ; ), mAb Huts-4 (Chemicon; ; ), and mAb 12G20 (Abcam; ; ); mouse antitalin mAb 8d4 (Sigma-Aldrich); and rabbit polyclonal abs antivinculin (V4139; Sigma-Aldrich) and anti–kindlin-2 (ab74030; Abcam).

Techniques: Labeling

Journal: The Journal of Cell Biology

Article Title: Active and inactive β1 integrins segregate into distinct nanoclusters in focal adhesions

doi: 10.1083/jcb.201707075

Figure Lengend Snippet: Distinct localization of active and inactive β1 integrins within FAs. (A) Representative confocal, STED, and STORM images of Hs578T cells colabeled with antiactive β1 integrin mAb 12G10 (OG488 or Cy3; Alexa Fluor 647) and antiinactive β1 integrin mAb AIIB2 (rhodamine or Alexa Fluor 405 through Alexa Fluor 647). White boxes indicate zoomed areas on the right. Bars: (main images) 5 µm; (insets) 2 µm. (B–D) Bars show mean Pearson’s correlation coefficients (± SD) obtained by confocal (B), STED (C), and STORM (D) images colabeled with mAb 12G10 and mAb AIIB2. Confocal, n = 10 images; STED, n = 20; STORM, n = 6. mAb 12G10 labelings targeted by two differently colored secondary abs were used as control for maximal colocalization. Confocal, n = 9 images; STED, n = 13; STORM, n = 6. (E) The three left bars show Hs578T cells labeled by mAbs AIIB2 ( n = 18 images), K20 ( n = 11), or 12G10 ( n = 13) targeted with two differently colored secondary abs each as controls for maximal colocalization. The remaining bars show mean Pearson’s correlation coefficients (± SD) of STED images of Hs578T cells colabeled by mAb 12G10 and mAb 9EG7 ( n = 12 images); mAb Huts-4 and mAb 9EG7 ( n = 11); mAb K20 and mAb 9EG7 ( n = 14); mAb K20 and mAb AIIB2 ( n = 17); mAb 12G10 and mAb AIIB2 ( n = 20); mAb 12G10 and mAb 13 ( n = 11); mAb Huts-4 and mAb AIIB2 ( n = 11); and mAb Huts-4 and mAb 13 ( n = 5). The individual colocalizations were statistically compared with the control with the lowest coefficient (12G10), providing the most stringent comparison. The mAb AIIB2 versus mAb12G10 combination was duplicated from C to facilitate direct comparison. (F) Bars show mean percentages ± SD of overlapping nanoclusters segmented in STED images of Hs578T cells colabeled with mAb 12G10 and mAb AIIB2 ( n = 20 images). Hs578T cells labeled using mAb K20 and two differently colored secondary abs both targeting mAb K20 were used as control for maximal overlap. n = 9 images. t test: *, P ≤ 0.05; ***, P ≤ 0.001.

Article Snippet: Primary abs used for immunostaining were rat anti–β1 integrin mAb 9EG7 (BD; ; ; ), mAb 13 (BD; ; ; ), and mAb AIIB2 (Developmental Studies Hybridoma Bank; ; ; ); mouse anti–β1 integrin mAb K20 (Santa Cruz Biotechnology, Inc.; ; ), mAb Huts-4 (Chemicon; ; ), and mAb 12G20 (Abcam; ; ); mouse antitalin mAb 8d4 (Sigma-Aldrich); and rabbit polyclonal abs antivinculin (V4139; Sigma-Aldrich) and anti–kindlin-2 (ab74030; Abcam).

Techniques: Labeling

Journal: The Journal of Cell Biology

Article Title: Active and inactive β1 integrins segregate into distinct nanoclusters in focal adhesions

doi: 10.1083/jcb.201707075

Figure Lengend Snippet: Anti–β1 integrin mAbs

Article Snippet: Primary abs used for immunostaining were rat anti–β1 integrin mAb 9EG7 (BD; ; ; ), mAb 13 (BD; ; ; ), and mAb AIIB2 (Developmental Studies Hybridoma Bank; ; ; ); mouse anti–β1 integrin mAb K20 (Santa Cruz Biotechnology, Inc.; ; ), mAb Huts-4 (Chemicon; ; ), and mAb 12G20 (Abcam; ; ); mouse antitalin mAb 8d4 (Sigma-Aldrich); and rabbit polyclonal abs antivinculin (V4139; Sigma-Aldrich) and anti–kindlin-2 (ab74030; Abcam).

Techniques:

Journal: The Journal of Cell Biology

Article Title: Active and inactive β1 integrins segregate into distinct nanoclusters in focal adhesions

doi: 10.1083/jcb.201707075

Figure Lengend Snippet: Nanoorganization of β1 integrins within FAs. (A) Representative images of an Hs578T cell labeled by anti–total β1 integrin mAb K20 (OG488) acquired by confocal microscopy (left) and STED (middle). The white box in the middle panel is zoomed in the right inset. (B) Representative intensity profiles along an individual adhesion (green/red lines in A; n = 10, one profile per image) reveal distinct intensity peaks by STED (red) but not by confocal microscopy (green). (C) Spatial distribution of identified intensity peaks within adhesions in the STED image from A. (D) Representative image of an Hs578T cell labeled with anti–β1 integrin mAb K20 (Alexa Fluor 405 through Alexa Fluor 647) acquired by TIRF (left) and STORM (middle). The white box is zoomed in on the right and shows clusters identified by DBSCAN. Bars: (main images) 5 µm; (insets) 500 nm.

Article Snippet: Primary abs used for immunostaining were rat anti–β1 integrin mAb 9EG7 (BD; ; ; ), mAb 13 (BD; ; ; ), and mAb AIIB2 (Developmental Studies Hybridoma Bank; ; ; ); mouse anti–β1 integrin mAb K20 (Santa Cruz Biotechnology, Inc.; ; ), mAb Huts-4 (Chemicon; ; ), and mAb 12G20 (Abcam; ; ); mouse antitalin mAb 8d4 (Sigma-Aldrich); and rabbit polyclonal abs antivinculin (V4139; Sigma-Aldrich) and anti–kindlin-2 (ab74030; Abcam).

Techniques: Labeling, Confocal Microscopy

Journal: The Journal of Cell Biology

Article Title: Active and inactive β1 integrins segregate into distinct nanoclusters in focal adhesions

doi: 10.1083/jcb.201707075

Figure Lengend Snippet: Distinct organization of active and inactive β1 integrin nanoclusters within FAs. (A) Left: Representative STED image of an Hs578T cell labeled with antiactive β1 integrin mAb 9EG7 (rhodamine). Right: Spatial distribution of detected intensity peaks within FAs. (B) Left: Representative STED image of Hs578T cells labeled with antiinactive β1 integrin mAb AIIB2 (rhodamine). Right: Spatial distribution of detected intensity peaks within FAs. (C) Bars show FAs scored for linear versus unstructured patterns in labelings of mAbs K20 ( n = 23 images), 9EG7 ( n = 21 images), and AIIB2 ( n = 18 images), displayed as mean percentages ± SEM. (D) Automated linearity analysis. Left: Means ± SD of fitted RANSAC lines needed to deplete all segmented clusters per FA for K20 ( n = 13 cells), 9EG7 ( n = 11 cells), and AIIB2 ( n = 11 cells) labelings. Right: Mean ± SD number of clusters per fitted RANSAC line for the same experimental set. Kolmogorov-Smirnov test: *, P < 0.05; **, P < 0.01. (E) Bars show median NND (quartile distribution in boxes; decile distributions in whiskers) of STED-identified clusters for mAb 9EG7 ( n = 19 images) and mAb AIIB2 ( n = 19) labelings within FAs. The two distributions display different variances. (F and G) Left: Representative STORM image of Hs578T cells labeled with mAb 9EG7 (F) or mAb AIIB2 (G; Alexa Fluor 405 through Alexa Fluor 647). White boxes indicate zooms shown on right. Red circles show DBSCAN-identified clusters in zoomed areas. Bars: (A, B, F, and G,main images) 5 µm; (F and G, insets) 500 nm. (H) Error bars show median NNDs (quartile distribution in boxes; decile distribution in whiskers) of β1 integrin clusters in STORM images of mAb 9EG7 ( n = 12 images) and mAb AIIB2 ( n = 14 images) labeling. The two distributions display different variances. (I and J) The bars show median cluster size (I) and median number of cluster localizations (J) of STORM images (quartile distribution in boxes; decile distribution in whiskers) of mAb 9EG7 ( n = 14 images) and mAb AIIB2 ( n = 14) labeling within FAs. t test: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Article Snippet: Primary abs used for immunostaining were rat anti–β1 integrin mAb 9EG7 (BD; ; ; ), mAb 13 (BD; ; ; ), and mAb AIIB2 (Developmental Studies Hybridoma Bank; ; ; ); mouse anti–β1 integrin mAb K20 (Santa Cruz Biotechnology, Inc.; ; ), mAb Huts-4 (Chemicon; ; ), and mAb 12G20 (Abcam; ; ); mouse antitalin mAb 8d4 (Sigma-Aldrich); and rabbit polyclonal abs antivinculin (V4139; Sigma-Aldrich) and anti–kindlin-2 (ab74030; Abcam).

Techniques: Labeling

Journal: The Journal of Cell Biology

Article Title: Active and inactive β1 integrins segregate into distinct nanoclusters in focal adhesions

doi: 10.1083/jcb.201707075

Figure Lengend Snippet: Distinct localization of active and inactive β1 integrins within FAs. (A) Representative confocal, STED, and STORM images of Hs578T cells colabeled with antiactive β1 integrin mAb 12G10 (OG488 or Cy3; Alexa Fluor 647) and antiinactive β1 integrin mAb AIIB2 (rhodamine or Alexa Fluor 405 through Alexa Fluor 647). White boxes indicate zoomed areas on the right. Bars: (main images) 5 µm; (insets) 2 µm. (B–D) Bars show mean Pearson’s correlation coefficients (± SD) obtained by confocal (B), STED (C), and STORM (D) images colabeled with mAb 12G10 and mAb AIIB2. Confocal, n = 10 images; STED, n = 20; STORM, n = 6. mAb 12G10 labelings targeted by two differently colored secondary abs were used as control for maximal colocalization. Confocal, n = 9 images; STED, n = 13; STORM, n = 6. (E) The three left bars show Hs578T cells labeled by mAbs AIIB2 ( n = 18 images), K20 ( n = 11), or 12G10 ( n = 13) targeted with two differently colored secondary abs each as controls for maximal colocalization. The remaining bars show mean Pearson’s correlation coefficients (± SD) of STED images of Hs578T cells colabeled by mAb 12G10 and mAb 9EG7 ( n = 12 images); mAb Huts-4 and mAb 9EG7 ( n = 11); mAb K20 and mAb 9EG7 ( n = 14); mAb K20 and mAb AIIB2 ( n = 17); mAb 12G10 and mAb AIIB2 ( n = 20); mAb 12G10 and mAb 13 ( n = 11); mAb Huts-4 and mAb AIIB2 ( n = 11); and mAb Huts-4 and mAb 13 ( n = 5). The individual colocalizations were statistically compared with the control with the lowest coefficient (12G10), providing the most stringent comparison. The mAb AIIB2 versus mAb12G10 combination was duplicated from C to facilitate direct comparison. (F) Bars show mean percentages ± SD of overlapping nanoclusters segmented in STED images of Hs578T cells colabeled with mAb 12G10 and mAb AIIB2 ( n = 20 images). Hs578T cells labeled using mAb K20 and two differently colored secondary abs both targeting mAb K20 were used as control for maximal overlap. n = 9 images. t test: *, P ≤ 0.05; ***, P ≤ 0.001.

Article Snippet: Primary abs used for immunostaining were rat anti–β1 integrin mAb 9EG7 (BD; ; ; ), mAb 13 (BD; ; ; ), and mAb AIIB2 (Developmental Studies Hybridoma Bank; ; ; ); mouse anti–β1 integrin mAb K20 (Santa Cruz Biotechnology, Inc.; ; ), mAb Huts-4 (Chemicon; ; ), and mAb 12G20 (Abcam; ; ); mouse antitalin mAb 8d4 (Sigma-Aldrich); and rabbit polyclonal abs antivinculin (V4139; Sigma-Aldrich) and anti–kindlin-2 (ab74030; Abcam).

Techniques: Control, Labeling, Comparison

Journal: Breast Cancer Research : BCR

Article Title: β1-integrin via NF-κB signaling is essential for acquisition of invasiveness in a model of radiation treated in situ breast cancer

doi: 10.1186/bcr3454

Figure Lengend Snippet: Phosphorylated-Akt up-regulated MCF10A cells form DCIS-like structures in three-dimensional lrECM cultures and in vivo . ( A ) MCF10A cells form acinar-like structures with hollow lumina when propagated in three-dimensional lrECM. When p-Akt is overexpressed (MCF10A-Akt), the colonies are significantly larger with cells filling the lumina. Phase-contrast micrographs and IF images stained with α6-integrin or p-Akt are shown. Bar = 10 μm. ( B ) The average colony size is increased in MCF10A-Akt compared to MCF10A. ( C ) Experimental schema of in vivo study. The MCF10A-Akt cells were injected intraductally into the mouse mammary duct and subsequently generated DCIS-like lesions. ( D ) H & E, IHC (β1-integrin, p-Akt and cleaved caspase-3) and IF (Ki-67) staining of intraductal xenografts. H & E stained image from the xenograft is almost identical to clinical human DCIS. Bar = 100 μm. DCIS, ductal carcinoma in situ ; IF, immunofluorescence; IHC, immunohistochemistry; lrECM, laminin-rich extracellular matrix.

Article Snippet: The following antibodies were used: anti-β1-integrin, clone 18 (BD Biosciences); anti-β1-integrin, AIIB2 (Sierra Biosource, Morgan Hill, CA, USA); anti-α5-integrin (Millipore); anti-α6-integrin, NKI-GoH3 (Millipore); anti-FN, IST-4 (Sigma); anti-FN EDA+ (Abcam, Cambridge, England); anti-cleaved caspase-3 (Cell signaling Technology); anti-NF-κB p65 (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan); anti-histone H1 (abcam) anti-β-actin (Sigma-Aldrich); ECL™ anti-mouse immunoglobulin G (IgG), HRP linked whole antibody (from sheep), NA931V (GE Healthcare, Buckinghamshire, UK); ECL™ anti-rabbit IgG, HRP linked whole antibody (from donkey), NA934V (GE Healthcare).

Techniques: In Vivo, Staining, Injection, Generated, In Situ, Immunofluorescence, Immunohistochemistry

Journal: Breast Cancer Research : BCR

Article Title: β1-integrin via NF-κB signaling is essential for acquisition of invasiveness in a model of radiation treated in situ breast cancer

doi: 10.1186/bcr3454

Figure Lengend Snippet: IR induces apoptosis in an active Akt-overexpressing model of human DCIS in three-dimensional lrECM . ( A ) Experimental schema. ( B ) IR-induced apoptosis was specifically observed in the luminal compartment of MCF10A-Akt structures. (Green = α6-integrin; red = cleaved caspase-3; blue = nuclei) Bar = 50 μm. ( C ) High content image analysis confirmed an increasing percentage of cells positive for cleaved caspase-3 with increasing IR doses. ( n = 200 acini, **, P < 1E-7) ( D ) Concentric measurements of mean intensity of cleaved caspase-3 showed significantly higher signal in the lumina of irradiated acini, compared to unirradiated controls. Dashed lines indicated edge of the acini. DCIS, ductal carcinoma in situ ; IR, ionizing radiation; lrECM, laminin-rich extracellular matrix.

Article Snippet: The following antibodies were used: anti-β1-integrin, clone 18 (BD Biosciences); anti-β1-integrin, AIIB2 (Sierra Biosource, Morgan Hill, CA, USA); anti-α5-integrin (Millipore); anti-α6-integrin, NKI-GoH3 (Millipore); anti-FN, IST-4 (Sigma); anti-FN EDA+ (Abcam, Cambridge, England); anti-cleaved caspase-3 (Cell signaling Technology); anti-NF-κB p65 (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan); anti-histone H1 (abcam) anti-β-actin (Sigma-Aldrich); ECL™ anti-mouse immunoglobulin G (IgG), HRP linked whole antibody (from sheep), NA931V (GE Healthcare, Buckinghamshire, UK); ECL™ anti-rabbit IgG, HRP linked whole antibody (from donkey), NA934V (GE Healthcare).

Techniques: Irradiation, In Situ

Journal: Breast Cancer Research : BCR

Article Title: β1-integrin via NF-κB signaling is essential for acquisition of invasiveness in a model of radiation treated in situ breast cancer

doi: 10.1186/bcr3454

Figure Lengend Snippet: An invasive phenotype emerged from a sub-population of cells surviving post-IR in three-dimensional lrECM . ( A ) Experimental schema of the recurrence model. At Day 12, cultures were exposed to Sham or 8 Gy IR. On Day 15, the colonies were taken out of three-dimensional lrECM, dissociated to make single cells, and expanded on two dimensional. Single cells were re-plated on three-dimensional lrECM and propagated until Day 30 (12 additional days). ( B ) Phase-contrast micrographs show that a distinct phenotype emerged by Day 30 of culture. Bar = 50 μm. IF images show α6-integrin or β1-integrin (green). Bar = 50 μm. ( C ) Invasive activity of MCF10A-Akt cells post-IR was quantified using invasion chambers. Graphical representation of the invasive cell numbers were normalized with control, non-irradiated cultures ( n = 3; **, P < 0.01). ( D ) Gelatin zymography shows that MMP-9 secretion was increased in culture medium of IR-treated MCF10A-Akt. ( E ) Matrix degradation activity was confirmed by fluorescently labeled DQ-gelatin matrix. Degraded gelatin is shown in green (22% ± 7 invasive cells versus 3% ± 1; n = 3; **, P < 0.01). DCIS, ductal carcinoma in situ ; IF, immunofluorescence; IR, ionizing radiation; lrECM, laminin-rich extracellular matrix; MMP-9, matrix metalloproteinase-9.

Article Snippet: The following antibodies were used: anti-β1-integrin, clone 18 (BD Biosciences); anti-β1-integrin, AIIB2 (Sierra Biosource, Morgan Hill, CA, USA); anti-α5-integrin (Millipore); anti-α6-integrin, NKI-GoH3 (Millipore); anti-FN, IST-4 (Sigma); anti-FN EDA+ (Abcam, Cambridge, England); anti-cleaved caspase-3 (Cell signaling Technology); anti-NF-κB p65 (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan); anti-histone H1 (abcam) anti-β-actin (Sigma-Aldrich); ECL™ anti-mouse immunoglobulin G (IgG), HRP linked whole antibody (from sheep), NA931V (GE Healthcare, Buckinghamshire, UK); ECL™ anti-rabbit IgG, HRP linked whole antibody (from donkey), NA934V (GE Healthcare).

Techniques: Activity Assay, Irradiation, Zymography, Labeling, In Situ, Immunofluorescence

Journal: Breast Cancer Research : BCR

Article Title: β1-integrin via NF-κB signaling is essential for acquisition of invasiveness in a model of radiation treated in situ breast cancer

doi: 10.1186/bcr3454

Figure Lengend Snippet: β1-integrin signaling is targeted to suppress invasive recurrence post-IR in MCF10A-Akt cells in three-dimensional lrECM . ( A ) On Day 30 cultures, up-regulated α5β1-integrin and down-regulated E-cadherin expression observed on the post-IR cultures. Columns, mean intensity ( n = 3; **, P < 0.01). ( B ) Up-regulation of FN and EDA+FN was observed in culture medium of cells post-IR. Columns, mean intensity ( n = 5; *, P < 0.05; ***, P < 0.001). ( C ) Phase-contrast images. IR-induced invasive phenotype was abrogated by AIIB2 compared to IgG. The antibodies were added from day 0 of the second three-dimensional cultures. Bar = 50 μm. ( D ) Apoptosis was measured by TUNEL-positive cells in AIIB2-treated cultures -/+ IR (mean = 18.1% ± 3.9, P < 0.01). ( E-F ) Matrigel chemoinvasion was significantly increased in the surviving cells post-IR, and inhibited by β1-integrin (AIIB2, five-fold, P < 0.01) or α5-integrin (P1D6, two-fold, P < 0.05) inhibitory antibodies. DCIS, ductal carcinoma in situ ; FN, fibronectin; IgG, immunoglobulin G; IR, ionizing radiation; lrECM, laminin-rich extracellular matrix; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Article Snippet: The following antibodies were used: anti-β1-integrin, clone 18 (BD Biosciences); anti-β1-integrin, AIIB2 (Sierra Biosource, Morgan Hill, CA, USA); anti-α5-integrin (Millipore); anti-α6-integrin, NKI-GoH3 (Millipore); anti-FN, IST-4 (Sigma); anti-FN EDA+ (Abcam, Cambridge, England); anti-cleaved caspase-3 (Cell signaling Technology); anti-NF-κB p65 (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan); anti-histone H1 (abcam) anti-β-actin (Sigma-Aldrich); ECL™ anti-mouse immunoglobulin G (IgG), HRP linked whole antibody (from sheep), NA931V (GE Healthcare, Buckinghamshire, UK); ECL™ anti-rabbit IgG, HRP linked whole antibody (from donkey), NA934V (GE Healthcare).

Techniques: Expressing, TUNEL Assay, In Situ, End Labeling

Journal: Breast Cancer Research : BCR

Article Title: β1-integrin via NF-κB signaling is essential for acquisition of invasiveness in a model of radiation treated in situ breast cancer

doi: 10.1186/bcr3454

Figure Lengend Snippet: Invasive phenotype emerged in a sub-population of irradiated MCF10A-Akt cells are associated with nuclear translocation of NF-κB . ( A ) On Day 30 cultures, immunoblotting of nuclear fraction shows up-regulated nuclear translocation of NF-κB p65 in the 8 Gy IR cultures. The intensities of NF-κB were normalized with nuclear protein, Histone H1 (**, P < 0.01, n = 4). ( B ) IF images of confocal microscopy show β1-integrin (green), NF-κB (red) and nuclei (blue). Bar = 50 μm. ( C ) The binding of NF-κB to the β1-integrin promoter region is up-regulated in MCF10A-Akt cells that survived after exposure to radiation (WT, wild-type, Mt, mutated oligonucleotide; n = 3, *, P < 0.05). ( D-E ) The NF-κB inhibitor, JSH-23, was added from day 0 of the second three-dimensional cultures. IR-induced chemoinvasion activity, which was inhibited by NF-κB inhibitor, JSH-23. Bar = 50 μm. IF, immunofluorescence; IR, ionizing radiation; NF-κB, nuclear factor-kappaB; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Article Snippet: The following antibodies were used: anti-β1-integrin, clone 18 (BD Biosciences); anti-β1-integrin, AIIB2 (Sierra Biosource, Morgan Hill, CA, USA); anti-α5-integrin (Millipore); anti-α6-integrin, NKI-GoH3 (Millipore); anti-FN, IST-4 (Sigma); anti-FN EDA+ (Abcam, Cambridge, England); anti-cleaved caspase-3 (Cell signaling Technology); anti-NF-κB p65 (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan); anti-histone H1 (abcam) anti-β-actin (Sigma-Aldrich); ECL™ anti-mouse immunoglobulin G (IgG), HRP linked whole antibody (from sheep), NA931V (GE Healthcare, Buckinghamshire, UK); ECL™ anti-rabbit IgG, HRP linked whole antibody (from donkey), NA934V (GE Healthcare).

Techniques: Irradiation, Translocation Assay, Western Blot, Confocal Microscopy, Binding Assay, Activity Assay, Immunofluorescence, TUNEL Assay, End Labeling

Journal: Journal of Cell Science

Article Title: E-cadherin-mediated force transduction signals regulate global cell mechanics

doi: 10.1242/jcs.185447

Figure Lengend Snippet: E-cadherin-based mechanotransduction alters cell traction and focal adhesions. (A) Illustration of the experimental setup combining magnetic twisting cytometry (MTC) and traction force microscopy (TFM). An oscillating magnetic field H generates a torque T, which displaces the magnetic beads. The amplitude of the bead displacement reflects the viscoelastic modulus of the bead–cell junction. Determined changes in cell stiffness or traction changes used cells with single beads, and excluded the majority of cells with multiple beads or beads at cell–cell contacts. (B) Time sequence of steps in combined MTC and TFM measurements. (C) Bar graph indicating changes in traction force (with or without load) exerted by MCF7 cells on collagen-coated polyacrylamide gels with elastic moduli of 8.8 kPa (−Load, n=7 cells; +Load, n=19 cells) and 34 kPa (−Load, n=11 cells, +Load, n=11 cells). (D) Bar graph showing changes in cell traction after force-loading beads modified with E-cadherin (E-cad, n=11 cells), poly-L-lysine (PLL, n=18 cells), neutral anti-E-cadherin antibody (Ntrl Ab, n=8 cells), or blocking anti-E-cadherin antibody (DECMA-1, n=8 cells). (E) Bar graph indicating traction changes (ΔRMS traction, Pa) after force-loading E-cadherin beads on cells adhered to collagen (n=11 cells), PLL (n=9 cells), or E-cadherin-coated polyacrylamide gels (34 kPa, n=7 cells). Results obtained with PLL-coated beads on cells adhered to PLL substrata are also shown (n=6 cells). With cells on either PLL- or E-cadherin-coated substrata, the medium contained integrin-blocking antibodies GOH3 and AIIB2. In C–E, the black bar denotes the same data used for statistical comparisons. Data presented are the mean±s.e.m. *P<0.01 (Student's t-test). Two or more independent experiments were performed. (F) Representative confocal immunofluorescence images of vinculin (green) and actin (gold) at the basal plane of cells on collagen-functionalized hydrogels. Cells were probed with E-cadherin (top) and DECMA-1 (bottom) functionalized beads, with (+Load) and without (−Load) 2 min of force loading. Scale bar: 10 µm.

Article Snippet: Integrins were blocked with anti-α6 antibody (GOH3) (20 μg/ml; Santa Cruz Biotechnology, SC-19622) and with anti-β1-integrin antibody AIIB2 (1:20 dilution) ( Barry et al., 2014 ).

Techniques: Cytometry, Microscopy, Magnetic Beads, Sequencing, Modification, Blocking Assay, Immunofluorescence

Journal: JCI Insight

Article Title: SGLT2 inhibitor dapagliflozin limits podocyte damage in proteinuric nondiabetic nephropathy

doi: 10.1172/jci.insight.98720

Figure Lengend Snippet: Representative images and quantification of immunofluorescence staining for F-actin (red) (A), α-actinin-4 (red) (B), and β1-integrin (red) (C) in podocytes exposed to control medium (control) or albumin (10 mg/ml), in the presence or absence of dapagliflozin (DAPA, 10 nM) for 6 hours. Nuclei were counterstained with DAPI (blue). Asterisks indicate podocytes with F-actin cytoskeletal remodeling. Quantifications were performed in 15 fields per sample. Data are the mean ± SEM (n = 4–6 samples for F-actin, n = 3 samples for α-actinin-4, n = 5 samples for β1-integrin) and were analyzed by ANOVA with Tukey’s post hoc test. Original magnification, ×630.

Article Snippet: For α-actinin-4 and β1-integrin staining, podocytes were incubated for 1 hour with rabbit anti–α-actinin-4 antibody (1:200, EPR2533(2), Origene) or with rat anti–β1-integrin antibody (undiluted, AIIB2, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa, USA) followed by a goat anti-rabbit Cy3-conjugated secondary antibody (1:80, 111-165-003, Jackson ImmunoResearch Laboratories).

Techniques: Immunofluorescence, Staining