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  • 99
    Thermo Fisher 4 6 diamidino 2 phenylindole dapi nuclear
    Landscapes of chromatin accessibility in the developing human retina and RO. ( A ) Schematic illustration of the overall experimental designs. Whole human neural retinae and ROs were collected for ATAC-seq (two replicates labeled with asterisk) and RNA-seq (inverted triangle). Development of human retinae and ROs was grouped into early, middle, and late stages and color coded. Immunostaining of GNAT1 in human retinae GW25 and ROs w30 is shown. Nuclei were stained with <t>4′,6-diamidino-2-phenylindole</t> <t>(DAPI).</t> Undiff, undifferentiated; Early, early developmental stage; Mid, middle developmental stage; Late, late developmental stage. Scale bars, 500 μm (bright-field images) and 10 μm (fluorescence images). PC1, principal component 1; PC2, principal component 2. ( B and C ) Immunostaining of RCVRN in human retinae (B) and ROs (C). Nuclei were stained with DAPI. NBL, neuroblastic layer; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars, 20 μm. ( D ) Heat map of Pearson correlations across all samples using all ATAC-seq peak signals. Relevant developmental stages are labeled with distinct colors as in (A). ( E ) PCA of chromatin accessibility during human retinal (blue) and RO (red) development in two dimensions. The black dotted arrow indicates the development process of retinogenesis. ( F and G ) Normalized epigenetic and expression profiles at the RCVRN loci during human retinal development (F) and RO differentiation (G). All signals were obtained from the University of California, Santa Cruz (UCSC) genome browser. ( H ) qRT-PCR analysis of the expression level of RCVRN ( n = 3) during RO differentiation. Data are means ± SEM. One-way analysis of variance (ANOVA) was performed. **** P
    4 6 Diamidino 2 Phenylindole Dapi Nuclear, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 276 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/4 6 diamidino 2 phenylindole dapi nuclear/product/Thermo Fisher
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    Millipore 4 6 diamidino 2 phenylindole dapi
    Immunohistochemical evaluation of the alcohol-induced inflammatory response. (A to F). TNF-α reactivity in the neural compartment in the CeA (A to C) and DVC (D to F) in control animals ( A, D; N = 4) and following chronic alcohol exposure ( B, E; N = 2) and 48 hours into withdrawal ( C, F; N = 2). Neurons are stained red with neuronal nuclear antigen (NeuN) and nuclei with <t>DAPI</t> shown in blue. The arrows in panels C. , E. and F. show the expression of TNF-α (green) surrounding cells expressing the neural marker NeuN. Dual labeling of a cell for both NeuN and TNF is shown in yellow. (G to L). ICAM-1 reactivity (green) in the endothelial compartment in the CeA (G to I) and DVC (J to L) , following chronic alcohol exposure (H, K) and 48 hours into withdrawal (I, L) . Endothelia are stained red with RECA-1 and nuclei with DAPI shown in blue. The arrow in panel H shows the expression of ICAM-1 by cells also expressing the endothelial cell marker RECA-1. Panels I and L have arrows showing small cells with a high degree of ICAM-1 staining found commonly in the chronic and withdrawal tissue that are absent in the control condition. Dual labeling of a cell for both RECA-1 and ICAM-1 is shown in yellow. CeA, central nucleus of the amygdale; DAPI, 5 μg/ml <t>4′-6-diamidino-2-phenylindole;</t> DVC, dorsal vagal complex; ICAM‐1, intercellular adhesion molecule 1;NeuN, neuronal nuclear antigen; RECA‐1, rat endothelial cell antigen‐1.
    4 6 Diamidino 2 Phenylindole Dapi, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12466 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/4 6 diamidino 2 phenylindole dapi/product/Millipore
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    99
    BioLegend 4 6 diamidino 2 phenylindole dapi
    Certolizumab pegol ( CZP ) induces rapid production of reactive oxygen species (ROS) and nuclear translocation of nuclear factor (erythroid-derived 2)-like ( Nrf2 ). Monocytes were incubated, or not, with CZP (5 μg/ml) and ROS production was assessed by H2DCFCA using flow cytometry. The time course of rapid ROS production over 30 minutes is shown in monocytes from four different healthy donors ( a ). Monocytes were incubated, or not, with CZP (5 μg/ml) for 90 minutes, and cellular localization of Nrf2 was analyzed by wide-field microscopy using an anti-Nrf2 antibody. <t>4',6-diamidino-2-phenylindole</t> ( <t>DAPI</t> ) was used as control for nucleus staining ( b ). Cytoplasmic and nuclear extracts were prepared after 90 minutes of cell culture and analyzed for Nrf2 detection ( c ). Time course analysis of Nrf2 translocation in nuclear fraction-enriched cell extracts of monocytes incubated, or not, with CZP (5 µg/ml) ( d ). TATA box binding protein ( TBP ) was used as control ( d ). Data are representative of four experiments ( b , c , d ). MFI geometric mean of fluorescence intensity
    4 6 Diamidino 2 Phenylindole Dapi, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/4 6 diamidino 2 phenylindole dapi/product/BioLegend
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    94
    Thermo Fisher 4 6 diamidino 2 phenylindole dapi
    PLK4 inhibition reverses centrosome amplification and cell hyperproliferation in AMPKα1-deleted MEFs. ( a ) PLK4 inhibition by Centrinone blocked centrosome amplification in AMPKα1 −/− MEFs. MEFs were treated with Centrinone (125 nM) or vehicle (DMSO) for 16 h. The cells were treated with nocodazole (20 ng/mL) for 4 hours to depolymerize the microtubules. Then, centrosomes, spindles, and DNA were co-stained with anti-γ-tubulin antibody (red), anti-α-tubulin antibody (green), and <t>4.6-diamidino-2-phenylindole</t> <t>(DAPI,</t> blue), respectively, and visualized by fluorescence microscope (scale bar = 20 μm); ( b ) PLK4 inhibition restrained cellular hyperproliferation in AMPKα1 −/− MEFs. MEF cells were seeded into 10-cm tissue culture dishes at 1 × 10 5 cells/dish. Centrinone was added at 125 nM. Every day, cells were harvested, counted using a Bio-Rad cell counter, and re-seeded into new dishes. Quantification data represent the mean ± S.D. from three separate experiments. * p
    4 6 Diamidino 2 Phenylindole Dapi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 34224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/4 6 diamidino 2 phenylindole dapi/product/Thermo Fisher
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    91
    Thermo Fisher dapi 4 6 diamidino 2 phenylindole dilactate
    MAP1889c induces DC activation via Toll-like receptor (TLR) 4 pathways. BMDCs derived from wild-type (WT), TLR2 –/– , and TLR4 –/– mice were treated with MAP1889c (10 μg/mL), LPS (100 ng/mL), or Pam3CSK4 (Pam3) (100 ng/mL) for 24 h. ( A ) MAP1889c-treated BMDCs for 1 h were fixed and then stained with <t>4’,6-diamidino-2-phenylindole</t> <t>(DAPI)</t> (blue) and Alexa Fluor-488-conjugated anti-MAP1889c antibody. Representative images out of three independent experiments are shown. Scale bar, 10 μm. ( B ) The cell lysates from BMDCs treated with MAP1889c for 6 h were used for immunoprecipitation with anti-mouse IgG and anti-His or with anti-TLR2 and anti-TLR4 antibodies. Thereafter, proteins were detected using immunoblotting with anti-His, anti-TLR2, or anti-TLR4 antibodies. The total is shown as the mean total cell lysates (input). ( C ) Production of IL-10, TNF-α, and IL-12p70 in the culture supernatants was determined by ELISA. All data are expressed as the mean ± SD ( n = 3). ( D ) Expression of CD80, CD86, and MHC class I molecules on BMDCs stimulated with each antigen was determined by staining and flow cytometry. The bar graphs show the mean percentage ± SEM of each surface molecule on CD11c + cells across three independent experiments. * p
    Dapi 4 6 Diamidino 2 Phenylindole Dilactate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 369 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Landscapes of chromatin accessibility in the developing human retina and RO. ( A ) Schematic illustration of the overall experimental designs. Whole human neural retinae and ROs were collected for ATAC-seq (two replicates labeled with asterisk) and RNA-seq (inverted triangle). Development of human retinae and ROs was grouped into early, middle, and late stages and color coded. Immunostaining of GNAT1 in human retinae GW25 and ROs w30 is shown. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Undiff, undifferentiated; Early, early developmental stage; Mid, middle developmental stage; Late, late developmental stage. Scale bars, 500 μm (bright-field images) and 10 μm (fluorescence images). PC1, principal component 1; PC2, principal component 2. ( B and C ) Immunostaining of RCVRN in human retinae (B) and ROs (C). Nuclei were stained with DAPI. NBL, neuroblastic layer; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars, 20 μm. ( D ) Heat map of Pearson correlations across all samples using all ATAC-seq peak signals. Relevant developmental stages are labeled with distinct colors as in (A). ( E ) PCA of chromatin accessibility during human retinal (blue) and RO (red) development in two dimensions. The black dotted arrow indicates the development process of retinogenesis. ( F and G ) Normalized epigenetic and expression profiles at the RCVRN loci during human retinal development (F) and RO differentiation (G). All signals were obtained from the University of California, Santa Cruz (UCSC) genome browser. ( H ) qRT-PCR analysis of the expression level of RCVRN ( n = 3) during RO differentiation. Data are means ± SEM. One-way analysis of variance (ANOVA) was performed. **** P

    Journal: Science Advances

    Article Title: Chromatin accessibility analysis reveals regulatory dynamics of developing human retina and hiPSC-derived retinal organoids

    doi: 10.1126/sciadv.aay5247

    Figure Lengend Snippet: Landscapes of chromatin accessibility in the developing human retina and RO. ( A ) Schematic illustration of the overall experimental designs. Whole human neural retinae and ROs were collected for ATAC-seq (two replicates labeled with asterisk) and RNA-seq (inverted triangle). Development of human retinae and ROs was grouped into early, middle, and late stages and color coded. Immunostaining of GNAT1 in human retinae GW25 and ROs w30 is shown. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Undiff, undifferentiated; Early, early developmental stage; Mid, middle developmental stage; Late, late developmental stage. Scale bars, 500 μm (bright-field images) and 10 μm (fluorescence images). PC1, principal component 1; PC2, principal component 2. ( B and C ) Immunostaining of RCVRN in human retinae (B) and ROs (C). Nuclei were stained with DAPI. NBL, neuroblastic layer; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars, 20 μm. ( D ) Heat map of Pearson correlations across all samples using all ATAC-seq peak signals. Relevant developmental stages are labeled with distinct colors as in (A). ( E ) PCA of chromatin accessibility during human retinal (blue) and RO (red) development in two dimensions. The black dotted arrow indicates the development process of retinogenesis. ( F and G ) Normalized epigenetic and expression profiles at the RCVRN loci during human retinal development (F) and RO differentiation (G). All signals were obtained from the University of California, Santa Cruz (UCSC) genome browser. ( H ) qRT-PCR analysis of the expression level of RCVRN ( n = 3) during RO differentiation. Data are means ± SEM. One-way analysis of variance (ANOVA) was performed. **** P

    Article Snippet: The slides were incubated in 4′,6-diamidino-2-phenylindole (DAPI) (1:1000; Thermo Fisher Scientific, D1306) in DPBS for 5 min, washed in DPBS (three times for 10 min), and cover slipped.

    Techniques: Labeling, RNA Sequencing Assay, Immunostaining, Staining, Fluorescence, Expressing, Quantitative RT-PCR

    Thrombogenicity and cell adhesion of gelatin hydrogel (GH). (A) Thrombogenicity of gelatin hydrogel (GH) was compared with that of collagen. In a 1500-s -1 blood stream, non-specific aggregation took place in 1 to 2 minutes, but no aggregation was induced after 3 minutes. In a 750-s -1 blood stream, GH induced no aggregation at all. Bars are 10 μm. (B) Cardiomyocytes (CM) were premixed with GH before transplantation. CM were stained with cardiac troponin-T and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). Most of the CM were entwined with GH, and they were distributed evenly. Bar is 100 μm.

    Journal: PLoS ONE

    Article Title: Gelatin Hydrogel Enhances the Engraftment of Transplanted Cardiomyocytes and Angiogenesis to Ameliorate Cardiac Function after Myocardial Infarction

    doi: 10.1371/journal.pone.0133308

    Figure Lengend Snippet: Thrombogenicity and cell adhesion of gelatin hydrogel (GH). (A) Thrombogenicity of gelatin hydrogel (GH) was compared with that of collagen. In a 1500-s -1 blood stream, non-specific aggregation took place in 1 to 2 minutes, but no aggregation was induced after 3 minutes. In a 750-s -1 blood stream, GH induced no aggregation at all. Bars are 10 μm. (B) Cardiomyocytes (CM) were premixed with GH before transplantation. CM were stained with cardiac troponin-T and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). Most of the CM were entwined with GH, and they were distributed evenly. Bar is 100 μm.

    Article Snippet: Nuclei were stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (Invitrogen).

    Techniques: Transplantation Assay, Staining

    Immunohistochemical evaluation of the alcohol-induced inflammatory response. (A to F). TNF-α reactivity in the neural compartment in the CeA (A to C) and DVC (D to F) in control animals ( A, D; N = 4) and following chronic alcohol exposure ( B, E; N = 2) and 48 hours into withdrawal ( C, F; N = 2). Neurons are stained red with neuronal nuclear antigen (NeuN) and nuclei with DAPI shown in blue. The arrows in panels C. , E. and F. show the expression of TNF-α (green) surrounding cells expressing the neural marker NeuN. Dual labeling of a cell for both NeuN and TNF is shown in yellow. (G to L). ICAM-1 reactivity (green) in the endothelial compartment in the CeA (G to I) and DVC (J to L) , following chronic alcohol exposure (H, K) and 48 hours into withdrawal (I, L) . Endothelia are stained red with RECA-1 and nuclei with DAPI shown in blue. The arrow in panel H shows the expression of ICAM-1 by cells also expressing the endothelial cell marker RECA-1. Panels I and L have arrows showing small cells with a high degree of ICAM-1 staining found commonly in the chronic and withdrawal tissue that are absent in the control condition. Dual labeling of a cell for both RECA-1 and ICAM-1 is shown in yellow. CeA, central nucleus of the amygdale; DAPI, 5 μg/ml 4′-6-diamidino-2-phenylindole; DVC, dorsal vagal complex; ICAM‐1, intercellular adhesion molecule 1;NeuN, neuronal nuclear antigen; RECA‐1, rat endothelial cell antigen‐1.

    Journal: Journal of Neuroinflammation

    Article Title: Temporal changes in innate immune signals in a rat model of alcohol withdrawal in emotional and cardiorespiratory homeostatic nuclei

    doi: 10.1186/1742-2094-9-97

    Figure Lengend Snippet: Immunohistochemical evaluation of the alcohol-induced inflammatory response. (A to F). TNF-α reactivity in the neural compartment in the CeA (A to C) and DVC (D to F) in control animals ( A, D; N = 4) and following chronic alcohol exposure ( B, E; N = 2) and 48 hours into withdrawal ( C, F; N = 2). Neurons are stained red with neuronal nuclear antigen (NeuN) and nuclei with DAPI shown in blue. The arrows in panels C. , E. and F. show the expression of TNF-α (green) surrounding cells expressing the neural marker NeuN. Dual labeling of a cell for both NeuN and TNF is shown in yellow. (G to L). ICAM-1 reactivity (green) in the endothelial compartment in the CeA (G to I) and DVC (J to L) , following chronic alcohol exposure (H, K) and 48 hours into withdrawal (I, L) . Endothelia are stained red with RECA-1 and nuclei with DAPI shown in blue. The arrow in panel H shows the expression of ICAM-1 by cells also expressing the endothelial cell marker RECA-1. Panels I and L have arrows showing small cells with a high degree of ICAM-1 staining found commonly in the chronic and withdrawal tissue that are absent in the control condition. Dual labeling of a cell for both RECA-1 and ICAM-1 is shown in yellow. CeA, central nucleus of the amygdale; DAPI, 5 μg/ml 4′-6-diamidino-2-phenylindole; DVC, dorsal vagal complex; ICAM‐1, intercellular adhesion molecule 1;NeuN, neuronal nuclear antigen; RECA‐1, rat endothelial cell antigen‐1.

    Article Snippet: Finally, for nuclei staining, the slides were washed with PBS, and then incubated with 5 μg/ml 4′-6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) at room temperature for five minutes.

    Techniques: Immunohistochemistry, Staining, Expressing, Marker, Labeling

    Certolizumab pegol ( CZP ) induces rapid production of reactive oxygen species (ROS) and nuclear translocation of nuclear factor (erythroid-derived 2)-like ( Nrf2 ). Monocytes were incubated, or not, with CZP (5 μg/ml) and ROS production was assessed by H2DCFCA using flow cytometry. The time course of rapid ROS production over 30 minutes is shown in monocytes from four different healthy donors ( a ). Monocytes were incubated, or not, with CZP (5 μg/ml) for 90 minutes, and cellular localization of Nrf2 was analyzed by wide-field microscopy using an anti-Nrf2 antibody. 4',6-diamidino-2-phenylindole ( DAPI ) was used as control for nucleus staining ( b ). Cytoplasmic and nuclear extracts were prepared after 90 minutes of cell culture and analyzed for Nrf2 detection ( c ). Time course analysis of Nrf2 translocation in nuclear fraction-enriched cell extracts of monocytes incubated, or not, with CZP (5 µg/ml) ( d ). TATA box binding protein ( TBP ) was used as control ( d ). Data are representative of four experiments ( b , c , d ). MFI geometric mean of fluorescence intensity

    Journal: Arthritis Research & Therapy

    Article Title: Anti-TNF certolizumab pegol induces antioxidant response in human monocytes via reverse signaling

    doi: 10.1186/s13075-016-0955-8

    Figure Lengend Snippet: Certolizumab pegol ( CZP ) induces rapid production of reactive oxygen species (ROS) and nuclear translocation of nuclear factor (erythroid-derived 2)-like ( Nrf2 ). Monocytes were incubated, or not, with CZP (5 μg/ml) and ROS production was assessed by H2DCFCA using flow cytometry. The time course of rapid ROS production over 30 minutes is shown in monocytes from four different healthy donors ( a ). Monocytes were incubated, or not, with CZP (5 μg/ml) for 90 minutes, and cellular localization of Nrf2 was analyzed by wide-field microscopy using an anti-Nrf2 antibody. 4',6-diamidino-2-phenylindole ( DAPI ) was used as control for nucleus staining ( b ). Cytoplasmic and nuclear extracts were prepared after 90 minutes of cell culture and analyzed for Nrf2 detection ( c ). Time course analysis of Nrf2 translocation in nuclear fraction-enriched cell extracts of monocytes incubated, or not, with CZP (5 µg/ml) ( d ). TATA box binding protein ( TBP ) was used as control ( d ). Data are representative of four experiments ( b , c , d ). MFI geometric mean of fluorescence intensity

    Article Snippet: The 4',6-diamidino-2-phenylindole (DAPI) was purchased from Biolegend (San Diego, CA, USA).

    Techniques: Translocation Assay, Derivative Assay, Incubation, Flow Cytometry, Cytometry, Microscopy, Staining, Cell Culture, Binding Assay, Fluorescence

    (a) Representative immunofluorescence staining of CD1a and programmed death ligand 2 (PD‐L2) in cutaneous squamous cell carcinoma (CSCC). Representative immunofluorescence staining on CSCC tissue frozen sections of case 31 evaluated by confocal microscopy. Dendritic cells are stained with CD1a (b, red), PD‐L2 (c, green) antibodies or 4′,6‐diamidino‐2‐phenylindole (DAPI) (a, blue), respectively. PD‐L2 + CD1a + cells are identified (d, yellow). (b) Representative immunofluorescence staining of CD83 and PD‐L2 in CSCC. Representative immunofluorescence staining on CSCC tissue frozen sections of case 31 evaluated by confocal microscopy. Dendritic cells are stained with CD83 (b, red), PD‐L2 (c, green) antibodies or DAPI (a, blue), respectively. PD‐L2 + CD83 + ]

    Journal: Clinical and Experimental Immunology

    Article Title: Programmed death‐1 ligands 1 and 2 expression in cutaneous squamous cell carcinoma and their relationship with tumour‐ infiltrating dendritic cells

    doi: 10.1111/cei.12921

    Figure Lengend Snippet: (a) Representative immunofluorescence staining of CD1a and programmed death ligand 2 (PD‐L2) in cutaneous squamous cell carcinoma (CSCC). Representative immunofluorescence staining on CSCC tissue frozen sections of case 31 evaluated by confocal microscopy. Dendritic cells are stained with CD1a (b, red), PD‐L2 (c, green) antibodies or 4′,6‐diamidino‐2‐phenylindole (DAPI) (a, blue), respectively. PD‐L2 + CD1a + cells are identified (d, yellow). (b) Representative immunofluorescence staining of CD83 and PD‐L2 in CSCC. Representative immunofluorescence staining on CSCC tissue frozen sections of case 31 evaluated by confocal microscopy. Dendritic cells are stained with CD83 (b, red), PD‐L2 (c, green) antibodies or DAPI (a, blue), respectively. PD‐L2 + CD83 + ]

    Article Snippet: Sections were mounted using Prolong Gold anti‐fade mounting medium with 4′,6‐diamidino‐2‐phenylindole [(DAPI) 422801; Biolegend, San Diego, CA, USA].

    Techniques: Immunofluorescence, Staining, Confocal Microscopy

    (a) Representative immunofluorescence staining of CD1a and programmed death ligand 1 (PD‐L1) in cutaneous squamous cell carcinoma (CSCC). Representative immunofluorescence staining on CSCC tissue frozen sections of case 30 evaluated by confocal microscopy. Dendritic cells are stained with CD1a (b, red), PD‐L1 (c, green) antibodies, or 4′,6‐diamidino‐2‐phenylindole (DAPI) (a, blue), respectively. PD‐L1 + CD1a + cells are identified (d, yellow). (b) Representative immunofluorescence staining of CD83 and PD‐L1 in CSCC. Representative immunofluorescence staining on CSCC tissue frozen sections of case 30 evaluated by confocal microscopy. Dendritic cells are stained with CD83 (b, red), PD‐L1 (c, green) antibodies or DAPI (a, blue), respectively. PD‐L1 + CD83 + ]

    Journal: Clinical and Experimental Immunology

    Article Title: Programmed death‐1 ligands 1 and 2 expression in cutaneous squamous cell carcinoma and their relationship with tumour‐ infiltrating dendritic cells

    doi: 10.1111/cei.12921

    Figure Lengend Snippet: (a) Representative immunofluorescence staining of CD1a and programmed death ligand 1 (PD‐L1) in cutaneous squamous cell carcinoma (CSCC). Representative immunofluorescence staining on CSCC tissue frozen sections of case 30 evaluated by confocal microscopy. Dendritic cells are stained with CD1a (b, red), PD‐L1 (c, green) antibodies, or 4′,6‐diamidino‐2‐phenylindole (DAPI) (a, blue), respectively. PD‐L1 + CD1a + cells are identified (d, yellow). (b) Representative immunofluorescence staining of CD83 and PD‐L1 in CSCC. Representative immunofluorescence staining on CSCC tissue frozen sections of case 30 evaluated by confocal microscopy. Dendritic cells are stained with CD83 (b, red), PD‐L1 (c, green) antibodies or DAPI (a, blue), respectively. PD‐L1 + CD83 + ]

    Article Snippet: Sections were mounted using Prolong Gold anti‐fade mounting medium with 4′,6‐diamidino‐2‐phenylindole [(DAPI) 422801; Biolegend, San Diego, CA, USA].

    Techniques: Immunofluorescence, Staining, Confocal Microscopy

    STAT4 deficiency promotes T cell immunosuppression and diminishes anti-tumoral response to HNSCC. (A) Gating strategy for determination of CD4 + and CD8 + T cell populations. (B) Percentage of CD4 + and CD8 + T cells in the draining lymph nodes and spleens of both tumor bearing and non tumor bearing WT and Stat4 −/− mice as determined by flow cytometric analysis. Significant differences between the tumor-bearing mice and non-tumor bearing mice of each mouse genotype are indicated by an * above the non-tumor bearing group plots. Data are presented as percentage of live cells. (C) Representative immunofluorescent images of CD3 + cells (green) in tumors of WT and Stat4 −/− mice. Tissues were counterstained with DAPI (blue). (D–I) Flow cytometric analysis showing intracellular expression of (D,E) TIM3, (F,G) PD-1, (H,I) CD69 by lymph node and splenic CD4 + and CD8 + populations of tumor bearing WT and Stat4 −/− mice. Representative dot plots and overlaid histogram plots of cellular expression of these immunosuppressive markers on gated CD4 + or CD8 + T cell populations are shown. Percentages of positive cells expressing these markers and mean fluorescent intensities (MFI) graphically represented to show differences between groups. Boxed data points are from mice showing lung metastases. (a) represents p

    Journal: Frontiers in Immunology

    Article Title: Immune Suppression Mediated by STAT4 Deficiency Promotes Lymphatic Metastasis in HNSCC

    doi: 10.3389/fimmu.2019.03095

    Figure Lengend Snippet: STAT4 deficiency promotes T cell immunosuppression and diminishes anti-tumoral response to HNSCC. (A) Gating strategy for determination of CD4 + and CD8 + T cell populations. (B) Percentage of CD4 + and CD8 + T cells in the draining lymph nodes and spleens of both tumor bearing and non tumor bearing WT and Stat4 −/− mice as determined by flow cytometric analysis. Significant differences between the tumor-bearing mice and non-tumor bearing mice of each mouse genotype are indicated by an * above the non-tumor bearing group plots. Data are presented as percentage of live cells. (C) Representative immunofluorescent images of CD3 + cells (green) in tumors of WT and Stat4 −/− mice. Tissues were counterstained with DAPI (blue). (D–I) Flow cytometric analysis showing intracellular expression of (D,E) TIM3, (F,G) PD-1, (H,I) CD69 by lymph node and splenic CD4 + and CD8 + populations of tumor bearing WT and Stat4 −/− mice. Representative dot plots and overlaid histogram plots of cellular expression of these immunosuppressive markers on gated CD4 + or CD8 + T cell populations are shown. Percentages of positive cells expressing these markers and mean fluorescent intensities (MFI) graphically represented to show differences between groups. Boxed data points are from mice showing lung metastases. (a) represents p

    Article Snippet: Samples were incubated with Alexa Fluor 488 conjugated goat anti hamster secondary antibody (Thermofisher Scientific, Foster City, CA, USA) at a concentration of 2 μg/mL for 1 h, then counterstained with DAPI (BioLegend, San Diego, CA).

    Techniques: Mouse Assay, Expressing

    PLK4 inhibition reverses centrosome amplification and cell hyperproliferation in AMPKα1-deleted MEFs. ( a ) PLK4 inhibition by Centrinone blocked centrosome amplification in AMPKα1 −/− MEFs. MEFs were treated with Centrinone (125 nM) or vehicle (DMSO) for 16 h. The cells were treated with nocodazole (20 ng/mL) for 4 hours to depolymerize the microtubules. Then, centrosomes, spindles, and DNA were co-stained with anti-γ-tubulin antibody (red), anti-α-tubulin antibody (green), and 4.6-diamidino-2-phenylindole (DAPI, blue), respectively, and visualized by fluorescence microscope (scale bar = 20 μm); ( b ) PLK4 inhibition restrained cellular hyperproliferation in AMPKα1 −/− MEFs. MEF cells were seeded into 10-cm tissue culture dishes at 1 × 10 5 cells/dish. Centrinone was added at 125 nM. Every day, cells were harvested, counted using a Bio-Rad cell counter, and re-seeded into new dishes. Quantification data represent the mean ± S.D. from three separate experiments. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Loss of AMPKalpha1 Triggers Centrosome Amplification via PLK4 Upregulation in Mouse Embryonic Fibroblasts

    doi: 10.3390/ijms21082772

    Figure Lengend Snippet: PLK4 inhibition reverses centrosome amplification and cell hyperproliferation in AMPKα1-deleted MEFs. ( a ) PLK4 inhibition by Centrinone blocked centrosome amplification in AMPKα1 −/− MEFs. MEFs were treated with Centrinone (125 nM) or vehicle (DMSO) for 16 h. The cells were treated with nocodazole (20 ng/mL) for 4 hours to depolymerize the microtubules. Then, centrosomes, spindles, and DNA were co-stained with anti-γ-tubulin antibody (red), anti-α-tubulin antibody (green), and 4.6-diamidino-2-phenylindole (DAPI, blue), respectively, and visualized by fluorescence microscope (scale bar = 20 μm); ( b ) PLK4 inhibition restrained cellular hyperproliferation in AMPKα1 −/− MEFs. MEF cells were seeded into 10-cm tissue culture dishes at 1 × 10 5 cells/dish. Centrinone was added at 125 nM. Every day, cells were harvested, counted using a Bio-Rad cell counter, and re-seeded into new dishes. Quantification data represent the mean ± S.D. from three separate experiments. * p

    Article Snippet: DNA was stained with antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA).

    Techniques: Inhibition, Amplification, Staining, Fluorescence, Microscopy

    AMPKα1 deletion confers centrosome amplification in MEFs. ( a ) Representative images of centrosome morphologies of WT and AMPKα1 −/− MEFs (scale bar = 20 μm) in the prometaphase of the cell cycle are shown. Centrosomes, spindles, and DNA were co-stained with anti-γ-tubulin antibody (red), anti-α-tubulin antibody (green), and 4.6-diamidino-2-phenylindole (DAPI, blue), respectively, and visualized by fluorescence microscope; ( b ) Quantification data for the percentage of MEFs containing > 2 centrosomes are presented. n = 5, * p

    Journal: International Journal of Molecular Sciences

    Article Title: Loss of AMPKalpha1 Triggers Centrosome Amplification via PLK4 Upregulation in Mouse Embryonic Fibroblasts

    doi: 10.3390/ijms21082772

    Figure Lengend Snippet: AMPKα1 deletion confers centrosome amplification in MEFs. ( a ) Representative images of centrosome morphologies of WT and AMPKα1 −/− MEFs (scale bar = 20 μm) in the prometaphase of the cell cycle are shown. Centrosomes, spindles, and DNA were co-stained with anti-γ-tubulin antibody (red), anti-α-tubulin antibody (green), and 4.6-diamidino-2-phenylindole (DAPI, blue), respectively, and visualized by fluorescence microscope; ( b ) Quantification data for the percentage of MEFs containing > 2 centrosomes are presented. n = 5, * p

    Article Snippet: DNA was stained with antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA).

    Techniques: Amplification, Staining, Fluorescence, Microscopy

    Disorganization of cardiac muscles and abnormalities of cardiomyocyte nuclei in Rrm1 KO hearts. A , images from the LVs of P7 hearts stained with phalloidin and DAPI. Scale bar , 20 μm. B , images from the LVs of P10 hearts stained with phalloidin and DAPI. Scale bar , 20 μm. C , quantification of the cardiomyocyte nuclear density in the samples in A and B . Data are shown as median values ( horizontal bars ) from two WT pups and two Rrm1 KO pups at P7 and from four WT pups and six Rrm1 KO pups at P10. The p values were computed with Mann–Whitney U tests: ns , nonsignificant. D , LVs of P7 or P14 hearts were stained with DAPI. Scale bar , 10 μm. E , proportions of DNA and RNA per total extracted nucleic acid from the hearts of P7, P9, and P15 pups. Data are shown as means ± S.E. ( error bars ) with n = 6–9. The p values were computed with an unpaired t test with Welch's correction: ****, p

    Journal: The Journal of Biological Chemistry

    Article Title: De novo dNTP production is essential for normal postnatal murine heart development

    doi: 10.1074/jbc.RA119.009492

    Figure Lengend Snippet: Disorganization of cardiac muscles and abnormalities of cardiomyocyte nuclei in Rrm1 KO hearts. A , images from the LVs of P7 hearts stained with phalloidin and DAPI. Scale bar , 20 μm. B , images from the LVs of P10 hearts stained with phalloidin and DAPI. Scale bar , 20 μm. C , quantification of the cardiomyocyte nuclear density in the samples in A and B . Data are shown as median values ( horizontal bars ) from two WT pups and two Rrm1 KO pups at P7 and from four WT pups and six Rrm1 KO pups at P10. The p values were computed with Mann–Whitney U tests: ns , nonsignificant. D , LVs of P7 or P14 hearts were stained with DAPI. Scale bar , 10 μm. E , proportions of DNA and RNA per total extracted nucleic acid from the hearts of P7, P9, and P15 pups. Data are shown as means ± S.E. ( error bars ) with n = 6–9. The p values were computed with an unpaired t test with Welch's correction: ****, p

    Article Snippet: The hearts were incubated in PBS containing 0.33 μ m rhodamine-phalloidin (Thermo Fisher Scientific) and 2 μg/ml DAPI (Thermo Fisher Scientific) overnight at 4 °C.

    Techniques: Staining

    MAP1889c induces DC activation via Toll-like receptor (TLR) 4 pathways. BMDCs derived from wild-type (WT), TLR2 –/– , and TLR4 –/– mice were treated with MAP1889c (10 μg/mL), LPS (100 ng/mL), or Pam3CSK4 (Pam3) (100 ng/mL) for 24 h. ( A ) MAP1889c-treated BMDCs for 1 h were fixed and then stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue) and Alexa Fluor-488-conjugated anti-MAP1889c antibody. Representative images out of three independent experiments are shown. Scale bar, 10 μm. ( B ) The cell lysates from BMDCs treated with MAP1889c for 6 h were used for immunoprecipitation with anti-mouse IgG and anti-His or with anti-TLR2 and anti-TLR4 antibodies. Thereafter, proteins were detected using immunoblotting with anti-His, anti-TLR2, or anti-TLR4 antibodies. The total is shown as the mean total cell lysates (input). ( C ) Production of IL-10, TNF-α, and IL-12p70 in the culture supernatants was determined by ELISA. All data are expressed as the mean ± SD ( n = 3). ( D ) Expression of CD80, CD86, and MHC class I molecules on BMDCs stimulated with each antigen was determined by staining and flow cytometry. The bar graphs show the mean percentage ± SEM of each surface molecule on CD11c + cells across three independent experiments. * p

    Journal: Cells

    Article Title: Mycobacterium avium subsp. paratuberculosis MAP1889c Protein Induces Maturation of Dendritic Cells and Drives Th2-Biased Immune Responses

    doi: 10.3390/cells9040944

    Figure Lengend Snippet: MAP1889c induces DC activation via Toll-like receptor (TLR) 4 pathways. BMDCs derived from wild-type (WT), TLR2 –/– , and TLR4 –/– mice were treated with MAP1889c (10 μg/mL), LPS (100 ng/mL), or Pam3CSK4 (Pam3) (100 ng/mL) for 24 h. ( A ) MAP1889c-treated BMDCs for 1 h were fixed and then stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue) and Alexa Fluor-488-conjugated anti-MAP1889c antibody. Representative images out of three independent experiments are shown. Scale bar, 10 μm. ( B ) The cell lysates from BMDCs treated with MAP1889c for 6 h were used for immunoprecipitation with anti-mouse IgG and anti-His or with anti-TLR2 and anti-TLR4 antibodies. Thereafter, proteins were detected using immunoblotting with anti-His, anti-TLR2, or anti-TLR4 antibodies. The total is shown as the mean total cell lysates (input). ( C ) Production of IL-10, TNF-α, and IL-12p70 in the culture supernatants was determined by ELISA. All data are expressed as the mean ± SD ( n = 3). ( D ) Expression of CD80, CD86, and MHC class I molecules on BMDCs stimulated with each antigen was determined by staining and flow cytometry. The bar graphs show the mean percentage ± SEM of each surface molecule on CD11c + cells across three independent experiments. * p

    Article Snippet: 4’,6-diamidino-2-phenylindole (DAPI) (D3571) and Texas Red® -X phalloidin (T7471) were obtained from Molecular Probes (Eugene, OR, USA).

    Techniques: Activation Assay, Derivative Assay, Mouse Assay, Staining, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry

    Visual confirmation of plasmid transfection using microscopy. Images were taken at 36 hours after microdermabrasion with multiple topical applications. ( a ) Multiphoton images showing fluorescent signals acquired from the green (EGFP-transfected cells) channel, red (tomato red showing all cell membranes) channel, and the combination. (i–iii) Topical application of the control vector. (iv–vi) Topical application of the EGFP plasmid. Yellow arrows indicate the hair follicles. Bars = 100 μm. ( b ) An image reconstructed from multiphoton Z -stacks showing the hair shafts with EGFP green fluorescence penetrating the dermis and epidermis. ( c ) Fluorescent images of frozen sections of the skin transfected with either EGFP or control vector. Cell nuclei are stained blue with DAPI. EGFP-transfected cells are stained green. Green staining is identified by arrows seen in hair follicles, hair shafts, the superficial epidermis, and more weakly, in the dermis. DAPI, 4',6-diamidino-2-phenylindole ; DC, dermal cell; E, epidermis; EGFP, enhanced green fluorescent protein; HF, hair follicle; HS, hair shaft. Bars = 100 μm.

    Journal: Molecular Therapy

    Article Title: Strengthening the Skin with Topical Delivery of Keratinocyte Growth Factor-1 Using a Novel DNA Plasmid

    doi: 10.1038/mt.2014.2

    Figure Lengend Snippet: Visual confirmation of plasmid transfection using microscopy. Images were taken at 36 hours after microdermabrasion with multiple topical applications. ( a ) Multiphoton images showing fluorescent signals acquired from the green (EGFP-transfected cells) channel, red (tomato red showing all cell membranes) channel, and the combination. (i–iii) Topical application of the control vector. (iv–vi) Topical application of the EGFP plasmid. Yellow arrows indicate the hair follicles. Bars = 100 μm. ( b ) An image reconstructed from multiphoton Z -stacks showing the hair shafts with EGFP green fluorescence penetrating the dermis and epidermis. ( c ) Fluorescent images of frozen sections of the skin transfected with either EGFP or control vector. Cell nuclei are stained blue with DAPI. EGFP-transfected cells are stained green. Green staining is identified by arrows seen in hair follicles, hair shafts, the superficial epidermis, and more weakly, in the dermis. DAPI, 4',6-diamidino-2-phenylindole ; DC, dermal cell; E, epidermis; EGFP, enhanced green fluorescent protein; HF, hair follicle; HS, hair shaft. Bars = 100 μm.

    Article Snippet: Nuclei were then stained with 4',6-diamidino-2-phenylindole blue (Invitrogen, Eugene, OR).

    Techniques: Plasmid Preparation, Transfection, Microscopy, Fluorescence, Staining

    CRN 83_152 causes re-localization of DNA within the nucleus . CRN-GFP fusion constructs were over expressed in N. benthamiana plants and imaged by confocal microscopy 48 h post-infiltration. (A) Leaves were co-infiltrated with RFP-fibrillarin and were DAPI stained by infiltrating with 4′,6-Diamidino-2-Phenylindole dilactate at a final concentration of 5 μg/ml. (B) N. benthamiana plants stably expressing histone RFP were infiltrated with CRN-GFP fusion constructs as described above. Scale bars = 5 μm.

    Journal: Frontiers in Plant Science

    Article Title: Characterization of cell death inducing Phytophthora capsici CRN effectors suggests diverse activities in the host nucleus

    doi: 10.3389/fpls.2013.00387

    Figure Lengend Snippet: CRN 83_152 causes re-localization of DNA within the nucleus . CRN-GFP fusion constructs were over expressed in N. benthamiana plants and imaged by confocal microscopy 48 h post-infiltration. (A) Leaves were co-infiltrated with RFP-fibrillarin and were DAPI stained by infiltrating with 4′,6-Diamidino-2-Phenylindole dilactate at a final concentration of 5 μg/ml. (B) N. benthamiana plants stably expressing histone RFP were infiltrated with CRN-GFP fusion constructs as described above. Scale bars = 5 μm.

    Article Snippet: For DAPI staining, leaves were infiltrated with 4′,6-Diamidino-2-Phenylindole dilactate (Invitrogen) at a final concentration of 5 μg/ml.

    Techniques: Construct, Confocal Microscopy, Staining, Concentration Assay, Stable Transfection, Expressing

    High glucose induces trunk level neural tube dysplasia. A: Quail embryos were exposed to high glucose for 3.5 days, sectioned transversely and stained with H E or immunofluorescently stained for Pax7. B, C: showing the representative appearance of the trunk neural tube in control and 25 mM glucose-treated embryos. D: Bar chart showing the incidence rate of NTDs increased with increases in glucose exposure. Pax7 is normally expressed in the dorsal side of the neural tube ( E ) but in the presence of high glucose Pax7 expression was inhibited ( F ). E’, F’: Sections counterstained with DAPI. Abbreviations: NT, neural tube. Scale bar = 50 µm.

    Journal: PLoS ONE

    Article Title: The Negative Influence of High-Glucose Ambience on Neurogenesis in Developing Quail Embryos

    doi: 10.1371/journal.pone.0066646

    Figure Lengend Snippet: High glucose induces trunk level neural tube dysplasia. A: Quail embryos were exposed to high glucose for 3.5 days, sectioned transversely and stained with H E or immunofluorescently stained for Pax7. B, C: showing the representative appearance of the trunk neural tube in control and 25 mM glucose-treated embryos. D: Bar chart showing the incidence rate of NTDs increased with increases in glucose exposure. Pax7 is normally expressed in the dorsal side of the neural tube ( E ) but in the presence of high glucose Pax7 expression was inhibited ( F ). E’, F’: Sections counterstained with DAPI. Abbreviations: NT, neural tube. Scale bar = 50 µm.

    Article Snippet: Lastly the specimens were counterstained with DAPI dye (1∶1000, Invitrogen, USA).

    Techniques: Staining, Expressing

    Cell autophagy against hypoxia leading to entosis. (A) Transmission electron microscope images show the ultrastructures of A431 and MCF-7 cells undergoing entosis. Right panels show greater details of left panels. Images show mitochondria swelling and DNA degradation of internalized cells at the early stage of cell-in-cell formation. (B) Images show internal cells completely losing their morphology with a large number of autophagic vacuoles and autophagic lysosomes at a later stage of cell-in-cell formation. (C) We used anticytochrome C antibody, which can only bind to cytochrome C released from mitochondria and indicates mitochondrial injury. Confocal images show strong fluorescence (green) in the cytoplasm of the internalized cells. Cells were labeled with CellTracker™ Red and cell nuclei were stained with DAPI. The scale bars are 10 µm. The right diagram shows the kinetics of cytochrome C release in the internalized tumor cells. (D) 2',7'-Dichlorofluorescin diacetate (DCFI-DA) was used to detect reactive oxygen species (ROS) in internalized cells of cell-in-cell structure. Confocal images show clear ROS (red) in the cytoplasm of internalized cells. Cells were labeled with CellTracker™ Green, and cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). The scale bars are 10 µm, which has been described in the legend in Figure 5 . The right diagram displayed the kinetics of ROS positive internalized cells in cell-in-cell structures.

    Journal: Journal of Breast Cancer

    Article Title: Cell-in-Cell Death Is Not Restricted by Caspase-3 Deficiency in MCF-7 Cells

    doi: 10.4048/jbc.2016.19.3.231

    Figure Lengend Snippet: Cell autophagy against hypoxia leading to entosis. (A) Transmission electron microscope images show the ultrastructures of A431 and MCF-7 cells undergoing entosis. Right panels show greater details of left panels. Images show mitochondria swelling and DNA degradation of internalized cells at the early stage of cell-in-cell formation. (B) Images show internal cells completely losing their morphology with a large number of autophagic vacuoles and autophagic lysosomes at a later stage of cell-in-cell formation. (C) We used anticytochrome C antibody, which can only bind to cytochrome C released from mitochondria and indicates mitochondrial injury. Confocal images show strong fluorescence (green) in the cytoplasm of the internalized cells. Cells were labeled with CellTracker™ Red and cell nuclei were stained with DAPI. The scale bars are 10 µm. The right diagram shows the kinetics of cytochrome C release in the internalized tumor cells. (D) 2',7'-Dichlorofluorescin diacetate (DCFI-DA) was used to detect reactive oxygen species (ROS) in internalized cells of cell-in-cell structure. Confocal images show clear ROS (red) in the cytoplasm of internalized cells. Cells were labeled with CellTracker™ Green, and cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). The scale bars are 10 µm, which has been described in the legend in Figure 5 . The right diagram displayed the kinetics of ROS positive internalized cells in cell-in-cell structures.

    Article Snippet: Coverslips were supported on slides using ProLong® Gold antifade reagent with 4',6'-diamidino-2-phenylindole (DAPI) reagent (Invitrogen) and mounted using Vectashield (Vector Laboratories, Burlingame, USA; Reactolab, Servion, Switzerland).

    Techniques: Transmission Assay, Microscopy, Fluorescence, Labeling, Staining

    Lysosome-dependent cell-in-cell death of A431 and MCF-7 cell lines. (A) Kinetic quantification of internalized terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) positive cells, internalized cells containing cleaved caspase-3 and internalized cells demonstrating lysosome activation in cell-in-cell structures of A431 cells and MCF-7 cells. One representative experiment of three independent experiments is shown. Data are presented as means±SD. (B) Confocal images show TUNEL positive (green) and DNA fragmentation (blue) of internalized A431 cells (red) and MCF-7 cells (red) at 48 hours. Both internalized A431 and MCF-7 showed inconspicuous changes in cell size and gradual nuclei degradation after entosis. Nuclei were labeled with 4',6-diamidino-2-phenylindole (DAPI). The scale bars are 10 µm. (C) As early as 6 hours after cell engulfment, the release of active cathepsin B (red) from the lysosomes into the plasma of the internalized cells was detected and was also seen in the surrounding cytoplasm of outer cells. Cells were stained with CellTracker™ Green and cell nuclei were labeled with DAPI. The scale bars are 10 µm. (D) A431 cells (green) and MCF-7 (green) cells are stained for lysosomes using LysoTracker™ Red (red), which binds to acidified compartments after 30 hours of culture. Confocal images show positive staining for cathepsin B in the cytoplasm of internalized cells. Scale bars are 10 µm.

    Journal: Journal of Breast Cancer

    Article Title: Cell-in-Cell Death Is Not Restricted by Caspase-3 Deficiency in MCF-7 Cells

    doi: 10.4048/jbc.2016.19.3.231

    Figure Lengend Snippet: Lysosome-dependent cell-in-cell death of A431 and MCF-7 cell lines. (A) Kinetic quantification of internalized terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) positive cells, internalized cells containing cleaved caspase-3 and internalized cells demonstrating lysosome activation in cell-in-cell structures of A431 cells and MCF-7 cells. One representative experiment of three independent experiments is shown. Data are presented as means±SD. (B) Confocal images show TUNEL positive (green) and DNA fragmentation (blue) of internalized A431 cells (red) and MCF-7 cells (red) at 48 hours. Both internalized A431 and MCF-7 showed inconspicuous changes in cell size and gradual nuclei degradation after entosis. Nuclei were labeled with 4',6-diamidino-2-phenylindole (DAPI). The scale bars are 10 µm. (C) As early as 6 hours after cell engulfment, the release of active cathepsin B (red) from the lysosomes into the plasma of the internalized cells was detected and was also seen in the surrounding cytoplasm of outer cells. Cells were stained with CellTracker™ Green and cell nuclei were labeled with DAPI. The scale bars are 10 µm. (D) A431 cells (green) and MCF-7 (green) cells are stained for lysosomes using LysoTracker™ Red (red), which binds to acidified compartments after 30 hours of culture. Confocal images show positive staining for cathepsin B in the cytoplasm of internalized cells. Scale bars are 10 µm.

    Article Snippet: Coverslips were supported on slides using ProLong® Gold antifade reagent with 4',6'-diamidino-2-phenylindole (DAPI) reagent (Invitrogen) and mounted using Vectashield (Vector Laboratories, Burlingame, USA; Reactolab, Servion, Switzerland).

    Techniques: End Labeling, TUNEL Assay, Activation Assay, Labeling, Staining

    Absence of caspase-3 protein in MCF-7 cells leading to an atypical apoptosis. (A) Expression of cleaved caspase-3 protein in A431 cells but not in MCF-7 cells. Both tumor cell lines were treated with (+) or without (–) staurosporine (staurosp.) for 16 hours and the cell lysates were analyzed by Western blotting. β-Actin was used as loading control. (B) Cytotoxicity assays of A431 and MCF-7 cells using the LDH method after treatment with staurosporine for 16 hours. Cells treated with the solvent dimethyl sulphoxide (DMSO) were used as negative controls. (C) Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay showed similar mortalities of the two cell lines following treatment with staurosporine for 16 hours. (D) Confocal images show positive TUNEL staining in both A431 and MCF-7 cells after treatment with staurosporine. Nuclear pyknosis was obvious in dying A431 cells but not in dying MCF-7 cells. Cells were labeled with CellTracker™ Red, and cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). The scale bars are 10 µm. (E) Cell cycle analysis of A431 and MCF-7 cells treated with or without staurosporine for 8 hours. The sub-G1 apoptotic peak demonstrating nuclear pyknosis in apoptotic cells is seen before the G0/G1 peak in A431 cells but not in MCF-7 cells after apoptosis induction. (F) DNA was prepared from cells untreated or treated for 8 hours or 16 hours with staurosporine and analyzed using a 1.6% agarose gel. There was obvious DNA ladder formation in A431 but not in MCF-7 cells after apoptosis induction. Lane M: DNA ladder.

    Journal: Journal of Breast Cancer

    Article Title: Cell-in-Cell Death Is Not Restricted by Caspase-3 Deficiency in MCF-7 Cells

    doi: 10.4048/jbc.2016.19.3.231

    Figure Lengend Snippet: Absence of caspase-3 protein in MCF-7 cells leading to an atypical apoptosis. (A) Expression of cleaved caspase-3 protein in A431 cells but not in MCF-7 cells. Both tumor cell lines were treated with (+) or without (–) staurosporine (staurosp.) for 16 hours and the cell lysates were analyzed by Western blotting. β-Actin was used as loading control. (B) Cytotoxicity assays of A431 and MCF-7 cells using the LDH method after treatment with staurosporine for 16 hours. Cells treated with the solvent dimethyl sulphoxide (DMSO) were used as negative controls. (C) Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay showed similar mortalities of the two cell lines following treatment with staurosporine for 16 hours. (D) Confocal images show positive TUNEL staining in both A431 and MCF-7 cells after treatment with staurosporine. Nuclear pyknosis was obvious in dying A431 cells but not in dying MCF-7 cells. Cells were labeled with CellTracker™ Red, and cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). The scale bars are 10 µm. (E) Cell cycle analysis of A431 and MCF-7 cells treated with or without staurosporine for 8 hours. The sub-G1 apoptotic peak demonstrating nuclear pyknosis in apoptotic cells is seen before the G0/G1 peak in A431 cells but not in MCF-7 cells after apoptosis induction. (F) DNA was prepared from cells untreated or treated for 8 hours or 16 hours with staurosporine and analyzed using a 1.6% agarose gel. There was obvious DNA ladder formation in A431 but not in MCF-7 cells after apoptosis induction. Lane M: DNA ladder.

    Article Snippet: Coverslips were supported on slides using ProLong® Gold antifade reagent with 4',6'-diamidino-2-phenylindole (DAPI) reagent (Invitrogen) and mounted using Vectashield (Vector Laboratories, Burlingame, USA; Reactolab, Servion, Switzerland).

    Techniques: Expressing, Western Blot, End Labeling, TUNEL Assay, Staining, Labeling, Cell Cycle Assay, Agarose Gel Electrophoresis

    Immunofluorescence staining of chondrocytes in the cryogel scaffolds in static or dynamic culture (frequency = 1 Hz) with different cyclic compressive-loading conditions. Blue: cell nucleus (DAPI); red: Col II (Cy 3); green: gelatin (FITC); scale bar = 100 µm. DAPI: 4′,6-diamidino-2-phenylindole; Cy 3: Cyanine 3; FITC: Fluorescein isothiocyanate.

    Journal: International Journal of Molecular Sciences

    Article Title: Effect of Cyclic Dynamic Compressive Loading on Chondrocytes and Adipose-Derived Stem Cells Co-Cultured in Highly Elastic Cryogel Scaffolds

    doi: 10.3390/ijms19020370

    Figure Lengend Snippet: Immunofluorescence staining of chondrocytes in the cryogel scaffolds in static or dynamic culture (frequency = 1 Hz) with different cyclic compressive-loading conditions. Blue: cell nucleus (DAPI); red: Col II (Cy 3); green: gelatin (FITC); scale bar = 100 µm. DAPI: 4′,6-diamidino-2-phenylindole; Cy 3: Cyanine 3; FITC: Fluorescein isothiocyanate.

    Article Snippet: Cell staining used 4,6-diamidino-2-phenylindole (DAPI) solution (Life Technologies, Carlsbad, CA, USA).

    Techniques: Immunofluorescence, Staining