hoechst 33342  (Millipore)

 
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    Name:
    bisBenzimide H 33342 trihydrochloride
    Description:

    Catalog Number:
    B2261
    Price:
    None
    Applications:
    Useful for staining DNA, chromosomes and nuclei. May be used for fluorescence microscopy or flow cytometry.Excitation max. = 346 nmEmission max. = 460 nm
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    Structured Review

    Millipore hoechst 33342
    bisBenzimide H 33342 trihydrochloride

    https://www.bioz.com/result/hoechst 33342/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hoechst 33342 - by Bioz Stars, 2021-04
    97/100 stars

    Images

    1) Product Images from "Protective Effect of γ-mangostin Isolated from the Peel of Garcinia mangostana against Glutamate-Induced Cytotoxicity in HT22 Hippocampal Neuronal Cells"

    Article Title: Protective Effect of γ-mangostin Isolated from the Peel of Garcinia mangostana against Glutamate-Induced Cytotoxicity in HT22 Hippocampal Neuronal Cells

    Journal: Biomolecules

    doi: 10.3390/biom11020170

    Effect of γ-mangostin (GM) on glutamate (Glu)-induced apoptotic cell death in HT22 cells. ( A ) Inhibitory effect of GM on Glu-induced chromatin condensation in HT22 cells. GM reduced Glu-induced annexin V-positive cells; annexin V-positive cells appear green. ( B ) The effect of GM on apoptosis was measured by fluorescence imaging using a Tali-Image-based cytometer. ( C ) Quantitative representation of the effect of GM on the proportion of apoptotic cells (annexin V-positive cells). Cells were treated with 5 mM Glu and 2.5 or 5 μM GM for 12 h and were then stained with Hoechst 33342. Images were obtained using a fluorescence microscope (20× magnification). Fluorescent images of cells stained using annexin V (green) and PI (red) and a merged image (bottom). The proportion of apoptotic cells was quantitatively analyzed to confirm the effect of GM (mean ± SEM, **  p
    Figure Legend Snippet: Effect of γ-mangostin (GM) on glutamate (Glu)-induced apoptotic cell death in HT22 cells. ( A ) Inhibitory effect of GM on Glu-induced chromatin condensation in HT22 cells. GM reduced Glu-induced annexin V-positive cells; annexin V-positive cells appear green. ( B ) The effect of GM on apoptosis was measured by fluorescence imaging using a Tali-Image-based cytometer. ( C ) Quantitative representation of the effect of GM on the proportion of apoptotic cells (annexin V-positive cells). Cells were treated with 5 mM Glu and 2.5 or 5 μM GM for 12 h and were then stained with Hoechst 33342. Images were obtained using a fluorescence microscope (20× magnification). Fluorescent images of cells stained using annexin V (green) and PI (red) and a merged image (bottom). The proportion of apoptotic cells was quantitatively analyzed to confirm the effect of GM (mean ± SEM, ** p

    Techniques Used: Fluorescence, Imaging, Cytometry, Staining, Microscopy

    2) Product Images from "A Single Amino Acid Mutation (R104P) in the E/DRY Motif of GPR40 Impairs Receptor Function"

    Article Title: A Single Amino Acid Mutation (R104P) in the E/DRY Motif of GPR40 Impairs Receptor Function

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0141303

    Effect of R104P mutant on cell surface localization of GPR40. (A) Translocation of GPR40 was studied with HEK293 cells transiently transfected with wild type or R104P mutant, and Cells were stained with non-permeabilized and permeabilized condition. GPR40 membranes were stained with myc-tagged GPR40 (red, arrows). Cell nuclei were stained with Hoechst 33342 (blue), 2 μg of DNA transfected. (B) Quantitative analysis of wild type and R104P mutant expression with plasmid of different amount as measured by membranes (M) and total protein (T), (shown in panel A). Data are means ± SEM (n = 3). *P
    Figure Legend Snippet: Effect of R104P mutant on cell surface localization of GPR40. (A) Translocation of GPR40 was studied with HEK293 cells transiently transfected with wild type or R104P mutant, and Cells were stained with non-permeabilized and permeabilized condition. GPR40 membranes were stained with myc-tagged GPR40 (red, arrows). Cell nuclei were stained with Hoechst 33342 (blue), 2 μg of DNA transfected. (B) Quantitative analysis of wild type and R104P mutant expression with plasmid of different amount as measured by membranes (M) and total protein (T), (shown in panel A). Data are means ± SEM (n = 3). *P

    Techniques Used: Mutagenesis, Translocation Assay, Transfection, Staining, Expressing, Plasmid Preparation

    3) Product Images from "The Anti-Cancer Effect of Linusorb B3 from Flaxseed Oil through the Promotion of Apoptosis, Inhibition of Actin Polymerization, and Suppression of Src Activity in Glioblastoma Cells"

    Article Title: The Anti-Cancer Effect of Linusorb B3 from Flaxseed Oil through the Promotion of Apoptosis, Inhibition of Actin Polymerization, and Suppression of Src Activity in Glioblastoma Cells

    Journal: Molecules

    doi: 10.3390/molecules25245881

    Inhibitory effect of LOB3 on actin polymerization by targeting Src and STAT3 in C6 cells. ( A ) Actin filaments (F-actin) and the nuclei of C6 cells treated with either LOB3 (30 μM) or CytoB (5 μM) for 12 h were stained with phalloidin and Hoechst 33342, respectively, and visualized under a confocal microscope. ( B ) Actin filaments (F-actin), caspase-3, and the nuclei of C6 cells treated with LOB3 (20 μM) for the indicated time were stained with phalloidin, caspase-3 antibody, and Hoechst 33342, respectively, and visualized under a confocal microscope. ( C ) Actin monomers were incubated with the indicated compounds for the indicated time, and actin polymerization was analyzed by an in vitro actin polymerization assay. ( D ) Actin filaments were incubated with the indicated compounds for the indicated time, and actin depolymerization was analyzed by an in vitro actin de-polymerization assay. ( E ) C6 cells were treated with the indicated doses of LOB3 for 12 h, and the protein levels of the phosphor and total forms of Src and STAT3 were determined by Western blot analysis. Results ( A – D ) are representative of three independent experiments. Statistical significance was analyzed by the Mann-Whitney U test. Data of band intensity ( F ) were measured and quantified using ImageJ. *  p
    Figure Legend Snippet: Inhibitory effect of LOB3 on actin polymerization by targeting Src and STAT3 in C6 cells. ( A ) Actin filaments (F-actin) and the nuclei of C6 cells treated with either LOB3 (30 μM) or CytoB (5 μM) for 12 h were stained with phalloidin and Hoechst 33342, respectively, and visualized under a confocal microscope. ( B ) Actin filaments (F-actin), caspase-3, and the nuclei of C6 cells treated with LOB3 (20 μM) for the indicated time were stained with phalloidin, caspase-3 antibody, and Hoechst 33342, respectively, and visualized under a confocal microscope. ( C ) Actin monomers were incubated with the indicated compounds for the indicated time, and actin polymerization was analyzed by an in vitro actin polymerization assay. ( D ) Actin filaments were incubated with the indicated compounds for the indicated time, and actin depolymerization was analyzed by an in vitro actin de-polymerization assay. ( E ) C6 cells were treated with the indicated doses of LOB3 for 12 h, and the protein levels of the phosphor and total forms of Src and STAT3 were determined by Western blot analysis. Results ( A – D ) are representative of three independent experiments. Statistical significance was analyzed by the Mann-Whitney U test. Data of band intensity ( F ) were measured and quantified using ImageJ. * p

    Techniques Used: Staining, Microscopy, Incubation, In Vitro, Polymerization Assay, Western Blot, MANN-WHITNEY

    Cytotoxic effect of LOB3 on C6 cells by apoptosis. ( A ) The nuclei of C6 cells treated with either staurosporine (2.5 μM) or LOB3 (20 and 30 μM) were stained with Hoechst 33342 and observed under a fluorescence microscope. Yellow arrows indicate nuclear shrinking and fragmentation. ( B ) C6 cells treated with the indicated doses of LOB3 for 24 h were stained with PI and annexin V-FITC, and the cell population was determined by flow cytometry analysis. ( C ) C6 cells were treated with the indicated doses of LOB3 for 24 h, and mRNA levels of Bcl-2, BAX, and p53 were analyzed by semiquantitative RT-PCR. ( D ) C6 cells were treated with either staurosporine (5 μM) or LOB3 (25 and 50 μM) for 24 h, and protein levels of pro-caspase-3, caspase-3, pro-caspase-9, and caspase-9 were determined by Western blot analysis. The data ( E , F ) are expressed as the means ± standard deviation (SD) of three experiments. Statistical significance was analyzed by the Mann-Whitney U test. Results ( A , B ). Data of band intensity ( E , F ) were measured and quantified using ImageJ. * p
    Figure Legend Snippet: Cytotoxic effect of LOB3 on C6 cells by apoptosis. ( A ) The nuclei of C6 cells treated with either staurosporine (2.5 μM) or LOB3 (20 and 30 μM) were stained with Hoechst 33342 and observed under a fluorescence microscope. Yellow arrows indicate nuclear shrinking and fragmentation. ( B ) C6 cells treated with the indicated doses of LOB3 for 24 h were stained with PI and annexin V-FITC, and the cell population was determined by flow cytometry analysis. ( C ) C6 cells were treated with the indicated doses of LOB3 for 24 h, and mRNA levels of Bcl-2, BAX, and p53 were analyzed by semiquantitative RT-PCR. ( D ) C6 cells were treated with either staurosporine (5 μM) or LOB3 (25 and 50 μM) for 24 h, and protein levels of pro-caspase-3, caspase-3, pro-caspase-9, and caspase-9 were determined by Western blot analysis. The data ( E , F ) are expressed as the means ± standard deviation (SD) of three experiments. Statistical significance was analyzed by the Mann-Whitney U test. Results ( A , B ). Data of band intensity ( E , F ) were measured and quantified using ImageJ. * p

    Techniques Used: Staining, Fluorescence, Microscopy, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Western Blot, Standard Deviation, MANN-WHITNEY

    4) Product Images from "Ovalitenone Inhibits the Migration of Lung Cancer Cells via the Suppression of AKT/mTOR and Epithelial-to-Mesenchymal Transition"

    Article Title: Ovalitenone Inhibits the Migration of Lung Cancer Cells via the Suppression of AKT/mTOR and Epithelial-to-Mesenchymal Transition

    Journal: Molecules

    doi: 10.3390/molecules26030638

    Effect of ovalitenone on cell viability and proliferation of lung cancer H460 and A549 cells. ( a ) Chemical structure of ovalitenone. ( b ) Cell viability of the cells was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazo-liumbromide (MTT) assay. ( c , d ) Effect of ovalitenone on cell proliferation. ( e , f ) The cells were treated with ovalitenone for 24, 48, and 72 h, and apoptotic and necrotic cells were evaluated by Hoechst 33342 and PI staining. ( g , h ) Annexin V/PI co-stained cells were examined using flow cytometry. ( i ) Cells were treated as indicated for 7 days, and colony was stained by crystal violet. All data are presented as mean ± SEM ( n = 3). * p
    Figure Legend Snippet: Effect of ovalitenone on cell viability and proliferation of lung cancer H460 and A549 cells. ( a ) Chemical structure of ovalitenone. ( b ) Cell viability of the cells was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazo-liumbromide (MTT) assay. ( c , d ) Effect of ovalitenone on cell proliferation. ( e , f ) The cells were treated with ovalitenone for 24, 48, and 72 h, and apoptotic and necrotic cells were evaluated by Hoechst 33342 and PI staining. ( g , h ) Annexin V/PI co-stained cells were examined using flow cytometry. ( i ) Cells were treated as indicated for 7 days, and colony was stained by crystal violet. All data are presented as mean ± SEM ( n = 3). * p

    Techniques Used: MTT Assay, Staining, Flow Cytometry

    Ovalitenone suppresses EMT. ( a , c , e ) H460 cells were treated with non-toxic concentrations of ovalitenone for 24 h. The cells were co-stained with anti-E-cadherin, N-cadherin, Snail antibodies, and Hoechst 33342. The expression of E-cadherin, N-cadherin, and Snail was examined using immunofluorescence. ( b , d , f ) The fluorescence intensity was analyzed by ImageJ software. Values represent the mean ± SEM. ( n  = 3). *  p
    Figure Legend Snippet: Ovalitenone suppresses EMT. ( a , c , e ) H460 cells were treated with non-toxic concentrations of ovalitenone for 24 h. The cells were co-stained with anti-E-cadherin, N-cadherin, Snail antibodies, and Hoechst 33342. The expression of E-cadherin, N-cadherin, and Snail was examined using immunofluorescence. ( b , d , f ) The fluorescence intensity was analyzed by ImageJ software. Values represent the mean ± SEM. ( n = 3). * p

    Techniques Used: Staining, Expressing, Immunofluorescence, Fluorescence, Software

    Ovalitenone suppresses cell migration, invasion and filopodia formation. ( a , b ) Cells were treated with ovalitenone for 24, 48, and 72 h, and migration activity was determined by wound healing assay. ( c ) Cell invasion was examined by transwell invasion assay. After 24 h, invading cells were stained with Hoechst 33342 and photographed. ( d , e ) Cells were treated with ovalitenone for 24 h, filopodia was stained with phalloidin-rhodamine, and the number of filopodia per cells was counted. All data are represented as mean ± SEM ( n = 3). * p
    Figure Legend Snippet: Ovalitenone suppresses cell migration, invasion and filopodia formation. ( a , b ) Cells were treated with ovalitenone for 24, 48, and 72 h, and migration activity was determined by wound healing assay. ( c ) Cell invasion was examined by transwell invasion assay. After 24 h, invading cells were stained with Hoechst 33342 and photographed. ( d , e ) Cells were treated with ovalitenone for 24 h, filopodia was stained with phalloidin-rhodamine, and the number of filopodia per cells was counted. All data are represented as mean ± SEM ( n = 3). * p

    Techniques Used: Migration, Activity Assay, Wound Healing Assay, Transwell Invasion Assay, Staining

    5) Product Images from "Concatenation of Human Connexin26 (hCx26) and Human Connexin46 (hCx46) for the Analysis of Heteromeric Gap Junction Hemichannels and Heterotypic Gap Junction Channels"

    Article Title: Concatenation of Human Connexin26 (hCx26) and Human Connexin46 (hCx46) for the Analysis of Heteromeric Gap Junction Hemichannels and Heterotypic Gap Junction Channels

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19092742

    Expression of the GFP-labeled monomeric and concatemeric connexins in HeLa cells. ( A ) Representative micrographs of cell pairs expressing GFP-labeled hCx46, hCx26, hCx46-hCx46, hCx26-hCx26, hCx46-hCx26, and hCx26-hCx46 are shown. The cells were imaged 24 h after transfection using a confocal laser scanning microscope. The nuclei (blue) were stained with Hoechst 33342. Gap junction plaques are indicated by arrows. Gap junction plaques were found in HeLa cells expressing hCx46, hCx26, and the four different tandems. In cells expressing the tandems, a trend to accumulate the proteins in intracellular organelles was observed. ( B ) Quantification of the gap junction plaque area formed by the monomers and the four different tandems in HeLa cells. The plaque area was calculated using the particle analyzer of ImageJ and normalized to the number of transfected cell pairs. At least three transfections were performed per construct. The results are given as average plaque area per cell pair [µm 2 ]. Error bars represent the SEM. The data were evaluated by a one-way ANOVA and a post-hoc Tukey test (**  p  ≤ 0.01, ***  p  ≤ 0.001) in comparison to hCx46 and hCx26 (###  p  ≤ 0.001). ( C ]. The data was normalized to the intensity of the hCx46 monomer.
    Figure Legend Snippet: Expression of the GFP-labeled monomeric and concatemeric connexins in HeLa cells. ( A ) Representative micrographs of cell pairs expressing GFP-labeled hCx46, hCx26, hCx46-hCx46, hCx26-hCx26, hCx46-hCx26, and hCx26-hCx46 are shown. The cells were imaged 24 h after transfection using a confocal laser scanning microscope. The nuclei (blue) were stained with Hoechst 33342. Gap junction plaques are indicated by arrows. Gap junction plaques were found in HeLa cells expressing hCx46, hCx26, and the four different tandems. In cells expressing the tandems, a trend to accumulate the proteins in intracellular organelles was observed. ( B ) Quantification of the gap junction plaque area formed by the monomers and the four different tandems in HeLa cells. The plaque area was calculated using the particle analyzer of ImageJ and normalized to the number of transfected cell pairs. At least three transfections were performed per construct. The results are given as average plaque area per cell pair [µm 2 ]. Error bars represent the SEM. The data were evaluated by a one-way ANOVA and a post-hoc Tukey test (** p ≤ 0.01, *** p ≤ 0.001) in comparison to hCx46 and hCx26 (### p ≤ 0.001). ( C ]. The data was normalized to the intensity of the hCx46 monomer.

    Techniques Used: Expressing, Labeling, Transfection, Laser-Scanning Microscopy, Staining, Construct

    6) Product Images from "Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging"

    Article Title: Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging

    Journal: Scientific Reports

    doi: 10.1038/srep27142

    In vivo MPM images of mouse hind limb skin. ( a ) 3D rendered hind limb skin images based on autofluorescence (AF) and moxifloxacin fluorescence. Cellular structures in the dermis were captured by approximately 4 times higher laser power (19 mW) than that used for moxifloxacin-treated skin (5 mW). ( b ) Quantification of epidermis and dermis fluorescence intensities. The junction in between the epidermis and dermis was divided along the basal cell layer. ( c ) MPM hind limb skin images at different depths based on topically treated moxifloxacin (green), Hoechst 33342 (blue), and intravenously injected tetramethylrhodamine (TAMRA, red) for identification of cells, their nuclei, and blood vessels, respectively. The stratum spinosum is shown in the first MIP MPM image (2–6 μm). Thin fibrous structures (yellow arrowhead) branching from dermal cell bodies (red arrowhead) and capillary endothelial cells (white arrowhead) with blood vessel are shown in subsequent MIP MPM images (32–40 μm and 48–60 μm, respectively). Scale bars, 50 μm. ( d ) Mobile cell tracking in time-lapse imaging of the dermis after topical treatment of moxifloxacin, in vivo . Mobile cell (orange arrowhead) passing by a static cell (black arrowhead) is shown. Total elapsed imaging time is 25 min during anesthesia of the mouse. Scale bars, 50 μm.
    Figure Legend Snippet: In vivo MPM images of mouse hind limb skin. ( a ) 3D rendered hind limb skin images based on autofluorescence (AF) and moxifloxacin fluorescence. Cellular structures in the dermis were captured by approximately 4 times higher laser power (19 mW) than that used for moxifloxacin-treated skin (5 mW). ( b ) Quantification of epidermis and dermis fluorescence intensities. The junction in between the epidermis and dermis was divided along the basal cell layer. ( c ) MPM hind limb skin images at different depths based on topically treated moxifloxacin (green), Hoechst 33342 (blue), and intravenously injected tetramethylrhodamine (TAMRA, red) for identification of cells, their nuclei, and blood vessels, respectively. The stratum spinosum is shown in the first MIP MPM image (2–6 μm). Thin fibrous structures (yellow arrowhead) branching from dermal cell bodies (red arrowhead) and capillary endothelial cells (white arrowhead) with blood vessel are shown in subsequent MIP MPM images (32–40 μm and 48–60 μm, respectively). Scale bars, 50 μm. ( d ) Mobile cell tracking in time-lapse imaging of the dermis after topical treatment of moxifloxacin, in vivo . Mobile cell (orange arrowhead) passing by a static cell (black arrowhead) is shown. Total elapsed imaging time is 25 min during anesthesia of the mouse. Scale bars, 50 μm.

    Techniques Used: In Vivo, Fluorescence, Injection, Cell Tracking Assay, Imaging

    7) Product Images from "Effect of methyl-beta-cyclodextrin on the viability and acrosome damage of sex-sorted sperm in frozen-thawed bovine semen"

    Article Title: Effect of methyl-beta-cyclodextrin on the viability and acrosome damage of sex-sorted sperm in frozen-thawed bovine semen

    Journal: Journal of Biological Research

    doi: 10.1186/s40709-016-0043-x

    Dot plot and histogram of X- and Y-chromosomes after Hoechst 33342 staining for sex sorting in frozen-thawed bovine sperm. Frozen-thawed sperm was automatically sorted into X- and Y-sperm populations by flow cytometry. The sex-sorted sperm based on differences of DNA contents. A dot plot is displaying Hoechst 33342 fluorescent intensity versus side-angle scatter (SSC, a ) and forward angle scatter (FSC, b ). Histogram is displaying Hoechst 33342 fluorescent intensity of sorted X- and Y-sperm ( c )
    Figure Legend Snippet: Dot plot and histogram of X- and Y-chromosomes after Hoechst 33342 staining for sex sorting in frozen-thawed bovine sperm. Frozen-thawed sperm was automatically sorted into X- and Y-sperm populations by flow cytometry. The sex-sorted sperm based on differences of DNA contents. A dot plot is displaying Hoechst 33342 fluorescent intensity versus side-angle scatter (SSC, a ) and forward angle scatter (FSC, b ). Histogram is displaying Hoechst 33342 fluorescent intensity of sorted X- and Y-sperm ( c )

    Techniques Used: Staining, Flow Cytometry, Cytometry

    8) Product Images from "Chemopreventive activity of celastrol in drug–resistant human colon carcinoma cell cultures"

    Article Title: Chemopreventive activity of celastrol in drug–resistant human colon carcinoma cell cultures

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25014

    The effect of celastrol on cell size of the SP subpopulation in LOVO/DX cell cultures (A) A gating strategy used to analyze only singlet viable cells. The cells were pre-incubated with celastrol for 5-10 minutes with subsequent incubation with DNA-binding dye Hoechst 33342 (Ho.) [5μg/ml] for 90 minutes at 37°C. Then the cells were stained with Annexin V-FITC and PI for exclusion of dead and apoptotic cells from the analysis. The cells-associated fluorescence was evaluated by the means of flow cytometry. SP (Side Population) is defined as subpopulation of cells that show the lowest Ho. content (a low-Ho.fluorescence “tail” in dual wavelength of fluorescence emission: Hoechst red [630nm]and Hoechst blue [455nm]). (B) Representative cytograms of cell size of the SP subpopulation in the presence of celastrol [20μM] or vehicle-DMSO. (C) Cell size of the SP subpopulation after treatment of LOVO/DX cultures with the range of celastrol concentrations. Results are expressed as E/E 0 ratios, where E = % of SP cells in cultures incubated with celastrol and E 0 = % of SP cells in cultures incubated with diluent -DMSO (mean ±SD, n=6 ; * p
    Figure Legend Snippet: The effect of celastrol on cell size of the SP subpopulation in LOVO/DX cell cultures (A) A gating strategy used to analyze only singlet viable cells. The cells were pre-incubated with celastrol for 5-10 minutes with subsequent incubation with DNA-binding dye Hoechst 33342 (Ho.) [5μg/ml] for 90 minutes at 37°C. Then the cells were stained with Annexin V-FITC and PI for exclusion of dead and apoptotic cells from the analysis. The cells-associated fluorescence was evaluated by the means of flow cytometry. SP (Side Population) is defined as subpopulation of cells that show the lowest Ho. content (a low-Ho.fluorescence “tail” in dual wavelength of fluorescence emission: Hoechst red [630nm]and Hoechst blue [455nm]). (B) Representative cytograms of cell size of the SP subpopulation in the presence of celastrol [20μM] or vehicle-DMSO. (C) Cell size of the SP subpopulation after treatment of LOVO/DX cultures with the range of celastrol concentrations. Results are expressed as E/E 0 ratios, where E = % of SP cells in cultures incubated with celastrol and E 0 = % of SP cells in cultures incubated with diluent -DMSO (mean ±SD, n=6 ; * p

    Techniques Used: Incubation, Binding Assay, Staining, Fluorescence, Flow Cytometry, Cytometry

    9) Product Images from "Tannic acid-modified silver nanoparticles for wound healing: the importance of size"

    Article Title: Tannic acid-modified silver nanoparticles for wound healing: the importance of size

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S154797

    Fibroblasts internalize AgNPs less effectively than monocytes. Notes:  Confocal microscopy images showing TA and unmodified AgNPs inside the cells (green). Nuclei were stained with Hoechst 33342. Images were captured using 63× objective lens and 1.5× digital zoom. ( A ) RAW control; ( B ) RAW TAm-13 nm; ( C ) RAW TAm-33 nm; ( D ) RAW TAm-46 nm; ( E ) RAW UN 10–65 nm; ( F ) L929 control; ( G ) L929 TAm-13 nm; ( H ) L929 TAm-33 nm; ( I ) L929 TAm-46 nm; ( J ) L929 UN 10–65 nm. Abbreviations:  AgNPs, silver nanoparticles; TAm, tannic acid-modified; UN, unmodified.
    Figure Legend Snippet: Fibroblasts internalize AgNPs less effectively than monocytes. Notes: Confocal microscopy images showing TA and unmodified AgNPs inside the cells (green). Nuclei were stained with Hoechst 33342. Images were captured using 63× objective lens and 1.5× digital zoom. ( A ) RAW control; ( B ) RAW TAm-13 nm; ( C ) RAW TAm-33 nm; ( D ) RAW TAm-46 nm; ( E ) RAW UN 10–65 nm; ( F ) L929 control; ( G ) L929 TAm-13 nm; ( H ) L929 TAm-33 nm; ( I ) L929 TAm-46 nm; ( J ) L929 UN 10–65 nm. Abbreviations: AgNPs, silver nanoparticles; TAm, tannic acid-modified; UN, unmodified.

    Techniques Used: Confocal Microscopy, Staining, Modification

    10) Product Images from "Positive effects of platelet-rich plasma (PRP) and a Sanguisorba officinalis polysaccharide on the proliferation and differentiation of anterior cruciate ligament (ACL) fibroblasts in vitro"

    Article Title: Positive effects of platelet-rich plasma (PRP) and a Sanguisorba officinalis polysaccharide on the proliferation and differentiation of anterior cruciate ligament (ACL) fibroblasts in vitro

    Journal: Pharmaceutical Biology

    doi: 10.1080/13880209.2020.1743325

    (A) Hoechst 33342 fluorescence staining of ACL fibroblasts after treatment with PRP, SOWPa (100 mg/kg) or PRP + SOWPa (100 mg/kg) for 72 h. (B) Flow cytometry analysis of ACL fibroblasts after treatment with PRP, SOWPa (100 mg/kg) or PRP + SOWPa (100 mg/kg) for 72 h.
    Figure Legend Snippet: (A) Hoechst 33342 fluorescence staining of ACL fibroblasts after treatment with PRP, SOWPa (100 mg/kg) or PRP + SOWPa (100 mg/kg) for 72 h. (B) Flow cytometry analysis of ACL fibroblasts after treatment with PRP, SOWPa (100 mg/kg) or PRP + SOWPa (100 mg/kg) for 72 h.

    Techniques Used: Fluorescence, Staining, Flow Cytometry

    11) Product Images from "Taperin (c9orf75), a mutated gene in nonsyndromic deafness, encodes a vertebrate specific, nuclear localized protein phosphatase one alpha (PP1?) docking protein"

    Article Title: Taperin (c9orf75), a mutated gene in nonsyndromic deafness, encodes a vertebrate specific, nuclear localized protein phosphatase one alpha (PP1?) docking protein

    Journal: Biology Open

    doi: 10.1242/bio.2011049

    Bimolecular fluorescence complementation (BiFC) demonstrates the in vivo interaction of taperin and PP1. The diagram depicts the design of the BiFC experiment, in which PP1γ was fused to an 84 amino acid C-terminal fragment of EYFP (pC/EYFP-PP1) and a targeting subunit fused to a 154 amino acid N-terminal fragment of EYFP (pN-EYFP-targeting subunit). If the proteins interact directly the fragments will complement each other to form a fluorescent EYFP molecule that can be excited with 513 nm light to emit light at 528 nm. As a positive control, U2OS cells were transfected with pNYFP-NIPP1 and pCYFP-PP1γ to demonstrate the nucleoplasmic interaction of these two proteins (green). When pNYFP-taperin was co-transfected with CYFP-PP1γ, a signal was detected throughout the cell (green), indicating interaction of the two proteins in both the nucleus and the cytoplasm. DNA is stained with Hoechst 33342 (blue). No signal was detected when NYFP-taperin KASA was co-transfected with CYFP-PP1γ (data not shown). Scale bars are 5 µM.
    Figure Legend Snippet: Bimolecular fluorescence complementation (BiFC) demonstrates the in vivo interaction of taperin and PP1. The diagram depicts the design of the BiFC experiment, in which PP1γ was fused to an 84 amino acid C-terminal fragment of EYFP (pC/EYFP-PP1) and a targeting subunit fused to a 154 amino acid N-terminal fragment of EYFP (pN-EYFP-targeting subunit). If the proteins interact directly the fragments will complement each other to form a fluorescent EYFP molecule that can be excited with 513 nm light to emit light at 528 nm. As a positive control, U2OS cells were transfected with pNYFP-NIPP1 and pCYFP-PP1γ to demonstrate the nucleoplasmic interaction of these two proteins (green). When pNYFP-taperin was co-transfected with CYFP-PP1γ, a signal was detected throughout the cell (green), indicating interaction of the two proteins in both the nucleus and the cytoplasm. DNA is stained with Hoechst 33342 (blue). No signal was detected when NYFP-taperin KASA was co-transfected with CYFP-PP1γ (data not shown). Scale bars are 5 µM.

    Techniques Used: Fluorescence, Bimolecular Fluorescence Complementation Assay, In Vivo, Positive Control, Transfection, Staining

    Taperin is recruited to sites of DNA damage  in vivo . (A)  Design of the experiment used to assess recruitment to discrete DNA lesions induced by UV laser microirradiation in pre-sensitized (by staining with Hoechst 33342) live cells. Following irradiation of a specific region of interest (ROI) the fluorescence intensity of GFP was then monitored in this same ROI over time.  (B)  Time-lapse imaging of GFP-taperin demonstrating recruitment of the fusion protein to the site of irradiation (arrow) over a 150 sec period. A pre-irradiation image was taken for comparison, and the first post-irradiation image was collected 0.002 sec after the laser fired.  (C)  The non-PP1 binding mutant GFP-taperin KASA  was subjected to the same treatment and demonstrated similar kinetics of recruitment to sites of UV-induced DNA damage.  (D)  As a positive control, the recruitment of PNUTS-GFP to DNA lesions was monitored over the same time scale.  (E)  The average % maximum fluorescence intensity ± SE was plotted versus time for GFP-Taperin (•,  n  = 10), GFP-Taperin KASA  (×,  n  = 10) and PNUTS-GFP (▴,  n  = 5). Data were normalized for photobleaching due to acquisition. The dashed line indicates the original fluorescence intensity within the ROI prior to DNA damage. Scale bars are 5 µm.
    Figure Legend Snippet: Taperin is recruited to sites of DNA damage in vivo . (A) Design of the experiment used to assess recruitment to discrete DNA lesions induced by UV laser microirradiation in pre-sensitized (by staining with Hoechst 33342) live cells. Following irradiation of a specific region of interest (ROI) the fluorescence intensity of GFP was then monitored in this same ROI over time. (B) Time-lapse imaging of GFP-taperin demonstrating recruitment of the fusion protein to the site of irradiation (arrow) over a 150 sec period. A pre-irradiation image was taken for comparison, and the first post-irradiation image was collected 0.002 sec after the laser fired. (C) The non-PP1 binding mutant GFP-taperin KASA was subjected to the same treatment and demonstrated similar kinetics of recruitment to sites of UV-induced DNA damage. (D) As a positive control, the recruitment of PNUTS-GFP to DNA lesions was monitored over the same time scale. (E) The average % maximum fluorescence intensity ± SE was plotted versus time for GFP-Taperin (•, n  = 10), GFP-Taperin KASA (×, n  = 10) and PNUTS-GFP (▴, n  = 5). Data were normalized for photobleaching due to acquisition. The dashed line indicates the original fluorescence intensity within the ROI prior to DNA damage. Scale bars are 5 µm.

    Techniques Used: In Vivo, Staining, Irradiation, Fluorescence, Imaging, Size-exclusion Chromatography, Binding Assay, Mutagenesis, Positive Control

    In vivo relocalization of PP1 by exogenously over-expressed taperin. (A) Transient overexpression of mCherry-tagged taperin (red) in HeLa EGFP-PP1γ cells relocalizes PP1γ (green) from nucleoli (arrow) and other nuclear sites of accumulation to the characteristic nucleoplasmic localization pattern of taperin itself. (B) As PP1α (green) exhibits a similar nucleoplasmic localization pattern to that of taperin, overexpression of mCherry-taperin (red) in HeLa EGFP-PP1α cells does not lead to an easily observable difference. (C) Over-expression of the non-PP1 binding mutant mCherry-taperin KASA does not lead to a relocalization of PP1γ in interphase HeLa EGFP-PP1γ cells. (D) Heterokaryon experiment in which HeLa cells transiently over-expressing mCherry-taperin (red) were fused to HeLa EGFP-PP1γ cells (green) in the presence of cycloheximide and imaged live 3 hours post-fusion. DNA was stained with Hoechst 33342 (blue). The dashed line indicates the cell membrane. The arrowhead indicates a nucleus containing both fusion proteins, in which PP1 is relocalized out of nucleoli (arrow) by taperin. (E) A similar experiment was carried out with the non-PP1 binding mutant mCherry-taperin KASA (red), which does not relocalize PP1 out of nucleoli (arrow) in nuclei containing both fusion proteins (arrowhead). Scale bars are 5 µM.
    Figure Legend Snippet: In vivo relocalization of PP1 by exogenously over-expressed taperin. (A) Transient overexpression of mCherry-tagged taperin (red) in HeLa EGFP-PP1γ cells relocalizes PP1γ (green) from nucleoli (arrow) and other nuclear sites of accumulation to the characteristic nucleoplasmic localization pattern of taperin itself. (B) As PP1α (green) exhibits a similar nucleoplasmic localization pattern to that of taperin, overexpression of mCherry-taperin (red) in HeLa EGFP-PP1α cells does not lead to an easily observable difference. (C) Over-expression of the non-PP1 binding mutant mCherry-taperin KASA does not lead to a relocalization of PP1γ in interphase HeLa EGFP-PP1γ cells. (D) Heterokaryon experiment in which HeLa cells transiently over-expressing mCherry-taperin (red) were fused to HeLa EGFP-PP1γ cells (green) in the presence of cycloheximide and imaged live 3 hours post-fusion. DNA was stained with Hoechst 33342 (blue). The dashed line indicates the cell membrane. The arrowhead indicates a nucleus containing both fusion proteins, in which PP1 is relocalized out of nucleoli (arrow) by taperin. (E) A similar experiment was carried out with the non-PP1 binding mutant mCherry-taperin KASA (red), which does not relocalize PP1 out of nucleoli (arrow) in nuclei containing both fusion proteins (arrowhead). Scale bars are 5 µM.

    Techniques Used: In Vivo, Over Expression, Binding Assay, Mutagenesis, Expressing, Staining

    Taperin can shuttle between the nucleus and cytosol. (A) In the heterokaryon approach depicted in the diagram, HeLa cells transiently expressing GFP-taperin (green) were mixed with non-transfected SW3T3 mouse cells. Cytoplasmic membranes were then fused by treatment with 50% PEG for 90 seconds and allowed to recover for 3 hours in media containing cycloheximide. DNA was then stained with Hoechst 33342 (blue) and cells imaged live to assess distribution of GFP-taperin between the original HeLa (arrowhead) and mouse (arrow) nuclei. The dashed line indicates the cell membrane. Scale bars are 5 µM. (B) Diagram depicting the FLIP experiment carried out to measure inter-compartmental dynamics of GFP-taperin, in which a cytoplasmic pool of the fusion protein is repeatedly bleached while the intensity of the nucleoplasmic pool is monitored over time. (C) Graph demonstrating the rapid loss of nucleoplasmic GFP (blue diamonds) when a cytoplasmic pool is bleached. The % maximum fluorescence intensity (normalized for photobleaching due to acquisition) is plotted versus the number of bleach events (each 100% laser power for a 0.05 sec duration). GFP-taperin (red squares) shows slower dynamics than free GFP, but a significant fraction of the nucleoplasmic pool is eventually bleached, indicating shuttling to the cytoplasm. (D) Sample dataset for each FLIP experiment. The lightning bolt indicates the photobleached region while the hashed circle shows the region of interest (ROI) that was monitored within the nucleus in each cell. To normalize for photobleaching caused by acquisition, a ROI was monitored in a neighboring cell. The number of bleach events is indicated in the bottom right corner. Each experiment was repeated 3 times. Scale bars are 5 µM.
    Figure Legend Snippet: Taperin can shuttle between the nucleus and cytosol. (A) In the heterokaryon approach depicted in the diagram, HeLa cells transiently expressing GFP-taperin (green) were mixed with non-transfected SW3T3 mouse cells. Cytoplasmic membranes were then fused by treatment with 50% PEG for 90 seconds and allowed to recover for 3 hours in media containing cycloheximide. DNA was then stained with Hoechst 33342 (blue) and cells imaged live to assess distribution of GFP-taperin between the original HeLa (arrowhead) and mouse (arrow) nuclei. The dashed line indicates the cell membrane. Scale bars are 5 µM. (B) Diagram depicting the FLIP experiment carried out to measure inter-compartmental dynamics of GFP-taperin, in which a cytoplasmic pool of the fusion protein is repeatedly bleached while the intensity of the nucleoplasmic pool is monitored over time. (C) Graph demonstrating the rapid loss of nucleoplasmic GFP (blue diamonds) when a cytoplasmic pool is bleached. The % maximum fluorescence intensity (normalized for photobleaching due to acquisition) is plotted versus the number of bleach events (each 100% laser power for a 0.05 sec duration). GFP-taperin (red squares) shows slower dynamics than free GFP, but a significant fraction of the nucleoplasmic pool is eventually bleached, indicating shuttling to the cytoplasm. (D) Sample dataset for each FLIP experiment. The lightning bolt indicates the photobleached region while the hashed circle shows the region of interest (ROI) that was monitored within the nucleus in each cell. To normalize for photobleaching caused by acquisition, a ROI was monitored in a neighboring cell. The number of bleach events is indicated in the bottom right corner. Each experiment was repeated 3 times. Scale bars are 5 µM.

    Techniques Used: Expressing, Transfection, Staining, Fluorescence, Size-exclusion Chromatography

    12) Product Images from "Concomitant epigenetic targeting of LSD1 and HDAC synergistically induces mitochondrial apoptosis in rhabdomyosarcoma cells"

    Article Title: Concomitant epigenetic targeting of LSD1 and HDAC synergistically induces mitochondrial apoptosis in rhabdomyosarcoma cells

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.239

    BMF, BIM and NOXA contribute to GSK690/JNJ-26481585-induced apoptosis. RD and RH30 were transiently transfected for 24 h with non-silencing siRNA or siRNA targeting BMF, BIM or NOXA mRNA. ( a, c,e ), BMF mRNA levels were assessed by qRT-PCR ( a ), BIM ( c ) and NOXA ( e ) protein levels were detected by Western blotting; β-Actin was used as loading control. ( b, d,f ), Cells were treated 24 h after transfection with 1 μ M GSK690 (RD cells) or 5 μ M GSK690 (RH30 cells) and/or 10 nM JNJ-26481585 for 72 h. Cell death was determined by flow cytometric analysis of DNA fragmentation of PI-stained nuclei (RD cells) or fluorescence-based microscope analysis of PI uptake using Hoechst 33342 and PI double staining (RH30 cells). In ( b, d,f ), mean and SD of three independent experiments performed in triplicate are shown; * P
    Figure Legend Snippet: BMF, BIM and NOXA contribute to GSK690/JNJ-26481585-induced apoptosis. RD and RH30 were transiently transfected for 24 h with non-silencing siRNA or siRNA targeting BMF, BIM or NOXA mRNA. ( a, c,e ), BMF mRNA levels were assessed by qRT-PCR ( a ), BIM ( c ) and NOXA ( e ) protein levels were detected by Western blotting; β-Actin was used as loading control. ( b, d,f ), Cells were treated 24 h after transfection with 1 μ M GSK690 (RD cells) or 5 μ M GSK690 (RH30 cells) and/or 10 nM JNJ-26481585 for 72 h. Cell death was determined by flow cytometric analysis of DNA fragmentation of PI-stained nuclei (RD cells) or fluorescence-based microscope analysis of PI uptake using Hoechst 33342 and PI double staining (RH30 cells). In ( b, d,f ), mean and SD of three independent experiments performed in triplicate are shown; * P

    Techniques Used: Transfection, Quantitative RT-PCR, Western Blot, Flow Cytometry, Staining, Fluorescence, Microscopy, Double Staining

    BAK contributes to GSK690/JNJ-26481585-induced apoptosis. RD and RH30 were transiently transfected for 48 h with non-silencing siRNA or siRNA targeting BAK. ( a ) Knockdown of BAK protein was detected by Western blotting, GAPDH served as loading control. ( b ) Cells were treated 48 h after transfection with 1 μ M GSK690 (RD cells) or 10 μ M GSK690 (RH30 cells) and/or 15 nM JNJ-26481585 for 72 h and cell death was determined by flow cytometric analysis of DNA fragmentation of PI-stained nuclei (RD cells) or fluorescence-based microscope analysis of PI uptake using Hoechst 33342 and PI double staining (RH30 cells). Mean and S.D. of three independent experiments performed in triplicate are shown; * P
    Figure Legend Snippet: BAK contributes to GSK690/JNJ-26481585-induced apoptosis. RD and RH30 were transiently transfected for 48 h with non-silencing siRNA or siRNA targeting BAK. ( a ) Knockdown of BAK protein was detected by Western blotting, GAPDH served as loading control. ( b ) Cells were treated 48 h after transfection with 1 μ M GSK690 (RD cells) or 10 μ M GSK690 (RH30 cells) and/or 15 nM JNJ-26481585 for 72 h and cell death was determined by flow cytometric analysis of DNA fragmentation of PI-stained nuclei (RD cells) or fluorescence-based microscope analysis of PI uptake using Hoechst 33342 and PI double staining (RH30 cells). Mean and S.D. of three independent experiments performed in triplicate are shown; * P

    Techniques Used: Transfection, Western Blot, Flow Cytometry, Staining, Fluorescence, Microscopy, Double Staining

    Overexpression of BCL-2 or MCL-1 reduces GSK690/JNJ-26481585-induced apoptosis. ( a ) RD and RH30 were transfected with empty vector (EV) or a vector containing a murine BCL-2 construct (mBCL-2). Expression of BCL-2 was assessed by Western blotting, asterisks indicate empty lanes. GAPDH was used as loading control. ( b ) Cells were treated with 1 μ M GSK690 (RD cells) or 5 μ M GSK690 (RH30 cells) and/or 15 nM JNJ-26481585 for 72 h. Cell death was determined by flow cytometric analysis of DNA fragmentation of PI-stained nuclei (RD cells) or fluorescence-based microscope analysis of PI uptake using Hoechst 33342 and PI double staining (RH30 cells). ( c ) Cells were treated with 1 μ M GSK690 (RD cells) or 10 μ M GSK690 (RH30 cells) and/or 15 nM JNJ-26481585 for 36 h (RD) or 24 h (RH30). Cell viability was assessed with MTT assay. ( d ) Cells were transfected with EV, wild-type MCL-1 (WT) or phospho-mutant MCL-1 (4A) constructs. Ectopic expression of MCL-1 constructs was detected by Western blotting, β-Actin served as loading control. ( e ) Transfected cells were treated with 1 μ M GSK690 (RD cells) or 5 μ M GSK690 (RH30 cells) and/or 15 nM JNJ-26481585 for 72 h. Cell death was determined by flow cytometric analysis of DNA fragmentation of PI-stained nuclei (RD cells) or fluorescence-based microscope analysis of PI uptake using Hoechst 33342 and PI double staining (RH30 cells). In ( b,c,e ) mean and S.D. of three independent experiments performed in triplicate are shown; * P
    Figure Legend Snippet: Overexpression of BCL-2 or MCL-1 reduces GSK690/JNJ-26481585-induced apoptosis. ( a ) RD and RH30 were transfected with empty vector (EV) or a vector containing a murine BCL-2 construct (mBCL-2). Expression of BCL-2 was assessed by Western blotting, asterisks indicate empty lanes. GAPDH was used as loading control. ( b ) Cells were treated with 1 μ M GSK690 (RD cells) or 5 μ M GSK690 (RH30 cells) and/or 15 nM JNJ-26481585 for 72 h. Cell death was determined by flow cytometric analysis of DNA fragmentation of PI-stained nuclei (RD cells) or fluorescence-based microscope analysis of PI uptake using Hoechst 33342 and PI double staining (RH30 cells). ( c ) Cells were treated with 1 μ M GSK690 (RD cells) or 10 μ M GSK690 (RH30 cells) and/or 15 nM JNJ-26481585 for 36 h (RD) or 24 h (RH30). Cell viability was assessed with MTT assay. ( d ) Cells were transfected with EV, wild-type MCL-1 (WT) or phospho-mutant MCL-1 (4A) constructs. Ectopic expression of MCL-1 constructs was detected by Western blotting, β-Actin served as loading control. ( e ) Transfected cells were treated with 1 μ M GSK690 (RD cells) or 5 μ M GSK690 (RH30 cells) and/or 15 nM JNJ-26481585 for 72 h. Cell death was determined by flow cytometric analysis of DNA fragmentation of PI-stained nuclei (RD cells) or fluorescence-based microscope analysis of PI uptake using Hoechst 33342 and PI double staining (RH30 cells). In ( b,c,e ) mean and S.D. of three independent experiments performed in triplicate are shown; * P

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Construct, Expressing, Western Blot, Flow Cytometry, Staining, Fluorescence, Microscopy, Double Staining, MTT Assay, Mutagenesis

    13) Product Images from "Shikonin overcomes drug resistance and induces necroptosis by regulating the miR-92a-1-5p/MLKL axis in chronic myeloid leukemia"

    Article Title: Shikonin overcomes drug resistance and induces necroptosis by regulating the miR-92a-1-5p/MLKL axis in chronic myeloid leukemia

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.103844

    Shikonin induces necroptosis in CML cells. ( A ) Results of MTT proliferation assays in K562, 32Dp210, and 32Dp210-T315I CML cells treated with 10 μΜ shikonin for 1.5 h ( i ) or 3 h ( ii ) following pre-treatment (1 h) with Nec-1 (50 μΜ) or zVAD-fmk (35 μΜ). Data are presented as the mean ± SD of three independent experiments. ( B ) Trypan blue exclusion assay results. CML cells were pretreated with or without 50 μΜ Nec-1 for 1 h and subsequently exposed to 10 μΜ shikonin for 3 h. The cells were then stained with trypan blue and the percentage of dead cells was determined under light microscopy. ( C ) Hoechst 33342/PI double staining was performed in CML cells preincubated with or without 50 μΜ Nec-1 and then treated with 0, 7.5, or 15 μΜ shikonin for 3 h. The percentage of PI-permeable cells in each group was determined by fluorescence microscopy. Blue fluorescence indicates staining with Hoechst 33342 and red fluorescence indicates PI staining. A few cells exhibited apoptotic characteristics (chromatin condensation and nuclear fragmentation), as indicated by white arrowheads. Magnification, 200×. ( D ) Electron microscopic examination of 32Dp210-T315I cells treated with 20 μM shikonin for 3 h revealed typical necrotic changes, including disorganization and loss (empty bubble-like formations) of cytoplasmic structures and plasma membrane rupture ( d , g , h ); syncytial nuclei with chromatin dissolution and disappearance of nucleoli ( e , f ); and severe damage to mitochondria with disruption of internal structures ( i ). Scale bars: 5 μm ( a , d ), 2 μm ( b , c , e , f , g ), 0.5 μm ( h ), and 0.2 μm ( i ). Quantification data are presented as the mean ± SD of three independent experiments. Representative results from three samples are shown. *p
    Figure Legend Snippet: Shikonin induces necroptosis in CML cells. ( A ) Results of MTT proliferation assays in K562, 32Dp210, and 32Dp210-T315I CML cells treated with 10 μΜ shikonin for 1.5 h ( i ) or 3 h ( ii ) following pre-treatment (1 h) with Nec-1 (50 μΜ) or zVAD-fmk (35 μΜ). Data are presented as the mean ± SD of three independent experiments. ( B ) Trypan blue exclusion assay results. CML cells were pretreated with or without 50 μΜ Nec-1 for 1 h and subsequently exposed to 10 μΜ shikonin for 3 h. The cells were then stained with trypan blue and the percentage of dead cells was determined under light microscopy. ( C ) Hoechst 33342/PI double staining was performed in CML cells preincubated with or without 50 μΜ Nec-1 and then treated with 0, 7.5, or 15 μΜ shikonin for 3 h. The percentage of PI-permeable cells in each group was determined by fluorescence microscopy. Blue fluorescence indicates staining with Hoechst 33342 and red fluorescence indicates PI staining. A few cells exhibited apoptotic characteristics (chromatin condensation and nuclear fragmentation), as indicated by white arrowheads. Magnification, 200×. ( D ) Electron microscopic examination of 32Dp210-T315I cells treated with 20 μM shikonin for 3 h revealed typical necrotic changes, including disorganization and loss (empty bubble-like formations) of cytoplasmic structures and plasma membrane rupture ( d , g , h ); syncytial nuclei with chromatin dissolution and disappearance of nucleoli ( e , f ); and severe damage to mitochondria with disruption of internal structures ( i ). Scale bars: 5 μm ( a , d ), 2 μm ( b , c , e , f , g ), 0.5 μm ( h ), and 0.2 μm ( i ). Quantification data are presented as the mean ± SD of three independent experiments. Representative results from three samples are shown. *p

    Techniques Used: MTT Assay, Trypan Blue Exclusion Assay, Staining, Light Microscopy, Double Staining, Fluorescence, Microscopy

    14) Product Images from "Developmentally arrested Austrofundulus limnaeus embryos have changes in post-translational modifications of histone H3"

    Article Title: Developmentally arrested Austrofundulus limnaeus embryos have changes in post-translational modifications of histone H3

    Journal: The Journal of Experimental Biology

    doi: 10.1242/jeb.131862

    PreDII and DII embryos display a similar distribution of H3K27me3.  Embryos were stained with anti-H3K27me3 and Hoechst 33342. The anterior tail region is shown for all images. Scale bar, 50 µm. The data represent at least 18 embryos (analysis of ≥6 embryos per trial,  N =3, for each embryo stage).
    Figure Legend Snippet: PreDII and DII embryos display a similar distribution of H3K27me3. Embryos were stained with anti-H3K27me3 and Hoechst 33342. The anterior tail region is shown for all images. Scale bar, 50 µm. The data represent at least 18 embryos (analysis of ≥6 embryos per trial, N =3, for each embryo stage).

    Techniques Used: Staining

    DII embryos stained with anti-H3K27me2 show an inner nuclear membrane localization pattern.  Embryos were stained with anti-H3K27me2 and Hoechst 33342. The anterior tail region is shown for all images. Scale bar, 50 µm. The data represent at least 18 embryos (analysis of ≥6 embryos per trial,  N =3, for each embryo stage).
    Figure Legend Snippet: DII embryos stained with anti-H3K27me2 show an inner nuclear membrane localization pattern. Embryos were stained with anti-H3K27me2 and Hoechst 33342. The anterior tail region is shown for all images. Scale bar, 50 µm. The data represent at least 18 embryos (analysis of ≥6 embryos per trial, N =3, for each embryo stage).

    Techniques Used: Staining

    The number of mitotic blastomeres decreases as embryos progress towards DII. Embryos were fixed and stained with anti-histone H3 serine 10 phosphate (H3S10P) to detect mitotic nuclei and Hoechst 33342 to stain DNA. (A) The anterior tail region is shown for all images: 18 dpf represents a preDII embryo, 24 dpf represents an embryo in early DII, and 32 dpf is a DII embryo. Scale bar, 50 µm. Representative embryos are shown (at least 18 embryos were analyzed; ≥6 embryos per trial, N =3, for each embryo stage). (B) The number of positive H3S10P cells was quantified in the tail region of embryos at various ages of preDII and DII. The data represent at least 18 embryos (analysis of ≥6 embryos per trial, N =3, for each embryo stage). The values are means±s.e.m. Identical letters indicate embryo groups with no significant difference; different letters indicate P
    Figure Legend Snippet: The number of mitotic blastomeres decreases as embryos progress towards DII. Embryos were fixed and stained with anti-histone H3 serine 10 phosphate (H3S10P) to detect mitotic nuclei and Hoechst 33342 to stain DNA. (A) The anterior tail region is shown for all images: 18 dpf represents a preDII embryo, 24 dpf represents an embryo in early DII, and 32 dpf is a DII embryo. Scale bar, 50 µm. Representative embryos are shown (at least 18 embryos were analyzed; ≥6 embryos per trial, N =3, for each embryo stage). (B) The number of positive H3S10P cells was quantified in the tail region of embryos at various ages of preDII and DII. The data represent at least 18 embryos (analysis of ≥6 embryos per trial, N =3, for each embryo stage). The values are means±s.e.m. Identical letters indicate embryo groups with no significant difference; different letters indicate P

    Techniques Used: Staining

    15) Product Images from "Valproate pretreatment protects pancreatic ?-cells from palmitate-induced ER stress and apoptosis by inhibiting glycogen synthase kinase-3?"

    Article Title: Valproate pretreatment protects pancreatic ?-cells from palmitate-induced ER stress and apoptosis by inhibiting glycogen synthase kinase-3?

    Journal: Journal of Biomedical Science

    doi: 10.1186/1423-0127-21-38

    Valproate pretreatment prevents palmitate - induced apoptosis and ER distension. INS-1 cells were pretreated with or without 1 mM VPA for 48 h before challenged with 0.25 mM PA. ( A ) After the treatments for 24 h, apoptosis was assessed by staining cells with Hoechst 33342/PI (a - c) , meanwhile the morphology of INS-1 cells was also captured (d - f) . Apoptosis was characterized by nucleus condensed or fragmented (red arrows) that intensely stained with Hoechst 33342 (blue fluorescence). In each case five to seven microscopic fields were photographed randomly. Scale bars: a-f = 400 × magnification. (B) After the treatments for 24 h or 48 h, the cells were stained with Annexin V and PI, and the percentage of apoptosis was detected by flow cytometry. Data are mean ± SD of three independent experiments. ** P
    Figure Legend Snippet: Valproate pretreatment prevents palmitate - induced apoptosis and ER distension. INS-1 cells were pretreated with or without 1 mM VPA for 48 h before challenged with 0.25 mM PA. ( A ) After the treatments for 24 h, apoptosis was assessed by staining cells with Hoechst 33342/PI (a - c) , meanwhile the morphology of INS-1 cells was also captured (d - f) . Apoptosis was characterized by nucleus condensed or fragmented (red arrows) that intensely stained with Hoechst 33342 (blue fluorescence). In each case five to seven microscopic fields were photographed randomly. Scale bars: a-f = 400 × magnification. (B) After the treatments for 24 h or 48 h, the cells were stained with Annexin V and PI, and the percentage of apoptosis was detected by flow cytometry. Data are mean ± SD of three independent experiments. ** P

    Techniques Used: Staining, Fluorescence, Flow Cytometry, Cytometry

    16) Product Images from "Detection of glioblastoma response to temozolomide combined with bevacizumab based on uMRI and uPET imaging reveals [18F]-fluoro-l-thymidine as an early and robust predictive marker for treatment efficacy"

    Article Title: Detection of glioblastoma response to temozolomide combined with bevacizumab based on uMRI and uPET imaging reveals [18F]-fluoro-l-thymidine as an early and robust predictive marker for treatment efficacy

    Journal: Neuro-Oncology

    doi: 10.1093/neuonc/nos260

    Early immunohistochemical determination of treatment effects on tumor vasculature and cell proliferation. (A) Representative images of RECA immunostaining (red) of 1 rat in each of the 4 different groups with Hoechst 33342 counterstaining (blue) for the
    Figure Legend Snippet: Early immunohistochemical determination of treatment effects on tumor vasculature and cell proliferation. (A) Representative images of RECA immunostaining (red) of 1 rat in each of the 4 different groups with Hoechst 33342 counterstaining (blue) for the

    Techniques Used: Immunohistochemistry, Immunostaining

    17) Product Images from "Ionizing radiation-induced foci persistence screen to discover enhancers of accelerated senescence"

    Article Title: Ionizing radiation-induced foci persistence screen to discover enhancers of accelerated senescence

    Journal: International journal of high throughput screening

    doi: 10.2147/IJHTS.S17076

    Optimizing quantification of development and resolution of ionizing radiation-induced foci (IRIF) in MCF7 Tet-On  green fluorescent protein (GFP)-IBD. A threshold of 1.1 μm was set for the minimum width for foci to exclude background. After a 48-hour induction with doxycycline, cells were pretreated for 1 hour with 6.25 μM etoposide and then dosed with 6 Gy IR. Twenty-four hours later, cells were scanned and imaged with the ImageXpress system.  A ) Automated imaging of GFP-labeled IRIF (pseudocolored green) in the leftmost panel, with Hoechst 33342-stained nuclei (pseudocolored red) superimposed in the middle panel. The right panel shows the result of automated segmentation of nuclei (Hoechst) and foci (GFP) prior to quantification by the granularity module of the MetaXpress software.  B ) Average number of IRIF per cell was calculated from four fields/well in triplicate wells at 2 hours (upper line, diamonds) and 24 hours (lower line, squares) at IR doses from 2 Gy to 14 Gy.  C ) The percentage of cells with nuclei with  > 20 foci counted at 2 hours (upper line, diamonds) and 24 hours (lower line, squares) at IR doses from 2 Gy to 14 Gy. Based on these data, 6 Gy was selected as a screening dose.
    Figure Legend Snippet: Optimizing quantification of development and resolution of ionizing radiation-induced foci (IRIF) in MCF7 Tet-On green fluorescent protein (GFP)-IBD. A threshold of 1.1 μm was set for the minimum width for foci to exclude background. After a 48-hour induction with doxycycline, cells were pretreated for 1 hour with 6.25 μM etoposide and then dosed with 6 Gy IR. Twenty-four hours later, cells were scanned and imaged with the ImageXpress system. A ) Automated imaging of GFP-labeled IRIF (pseudocolored green) in the leftmost panel, with Hoechst 33342-stained nuclei (pseudocolored red) superimposed in the middle panel. The right panel shows the result of automated segmentation of nuclei (Hoechst) and foci (GFP) prior to quantification by the granularity module of the MetaXpress software. B ) Average number of IRIF per cell was calculated from four fields/well in triplicate wells at 2 hours (upper line, diamonds) and 24 hours (lower line, squares) at IR doses from 2 Gy to 14 Gy. C ) The percentage of cells with nuclei with > 20 foci counted at 2 hours (upper line, diamonds) and 24 hours (lower line, squares) at IR doses from 2 Gy to 14 Gy. Based on these data, 6 Gy was selected as a screening dose.

    Techniques Used: Imaging, Labeling, Staining, Software

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    Article Title: Development of an in vitro senescent hepatic cell model for metabolic studies in aging
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    Article Snippet: A rabbit polyclonal antibody CXCR4 (1:200, Abcam, Hong Kong, China) was used for cellular fluorescence labeling. .. Hoechst 33342 (2 µg/ml, Sigma, USA) was used for staining cell nuclei. .. The rest cells were labeled with bromodeoxyuridine (5-bromo-2-deoxyuridine [BrdU], 30 μg/ml, Sigma, St. Louis, MO, USA) for 3 days before intravenous administration to identify the cell migration in the ischemic brain, and then the labeled cells were washed and digested for transplantation.

    Incubation:

    Article Title: Identification and characterization of a resident vascular stem/progenitor cell population in preexisting blood vessels
    Article Snippet: Hoechst staining was performed as described previously ( ). .. Briefly, cell-surface antigen staining was performed and cell suspensions were incubated with Hoechst 33342 (5 μg/ml) (Sigma) at 37°C for 90 min in DMEM (Sigma) (2% fetal calf serum, 1 mM HEPES) at a concentration of 1 × 106 nucleated cells/ml in the presence or absence of verapamil (50 μmol/l, Sigma). .. To analyse the cell-cycle status by Pyronin Y (PY) staining, cells were first stained with Hoechst 33342 at 37°C.

    Article Title: Histone demethylase Lsd1 represses hematopoietic stem and progenitor cell signatures during blood cell maturation
    Article Snippet: Donor contribution in peripheral blood, mature bone marrow lineage, bone marrow stem cell and progenitor cell compartment, and spleen were measured by cell surface staining for the CD45.1 and CD45.2 markers as well as genomic DNA analysis of sorted populations. .. For cell cycle analysis, cells were resuspended in prewarmed DMEM +2% FCS at a concentration 106 /ml followed by a 1-hr incubation at 37°C with 4 µg/ml Hoechst 33342 (Sigma, St Louis, MO). ..

    Article Title: Development of a Three-Dimensional In Vitro Model for Longitudinal Observation of Cell Behavior: Monitoring by Magnetic Resonance Imaging and Optical Imaging
    Article Snippet: For anti-vimentin antibody detection, the secondary antibody anti-mouse 488 (Thermo Fisher Scientific) was used. .. Nuclei were detected with the bisbenzimide Hoechst Dye 33342 (1:1,000, Sigma Aldrich) or with Sytox green (1:1,000, Invitrogen) after 10 min incubation. .. The glass cover slips were embedded with Aquamount (Polyscience Inc., Eppelheim, Germany) and further processed.

    Concentration Assay:

    Article Title: Identification and characterization of a resident vascular stem/progenitor cell population in preexisting blood vessels
    Article Snippet: Hoechst staining was performed as described previously ( ). .. Briefly, cell-surface antigen staining was performed and cell suspensions were incubated with Hoechst 33342 (5 μg/ml) (Sigma) at 37°C for 90 min in DMEM (Sigma) (2% fetal calf serum, 1 mM HEPES) at a concentration of 1 × 106 nucleated cells/ml in the presence or absence of verapamil (50 μmol/l, Sigma). .. To analyse the cell-cycle status by Pyronin Y (PY) staining, cells were first stained with Hoechst 33342 at 37°C.

    Article Title: 18F-misonidazole PET imaging of hypoxia in micrometastases and macroscopic xenografts of human non-small cell lung cancer: a correlation with autoradiography and histological findings
    Article Snippet: The proliferation marker, bromodeoxyuridine (Roche Diagnostics, Indianapolis, IN) was first dissolved in dimethyl sulfoxide and further diluted in physiological saline to a final concentration of 20 mg/ml. .. The blood perfusion marker, Hoechst 33342 (Sigma-Aldrich, St. Louis, MO) was dissolved in physiological saline at a concentration of 5 mg/ml. .. In all cases, fresh drug solutions were prepared on the day of injection.

    Article Title: Histone demethylase Lsd1 represses hematopoietic stem and progenitor cell signatures during blood cell maturation
    Article Snippet: Donor contribution in peripheral blood, mature bone marrow lineage, bone marrow stem cell and progenitor cell compartment, and spleen were measured by cell surface staining for the CD45.1 and CD45.2 markers as well as genomic DNA analysis of sorted populations. .. For cell cycle analysis, cells were resuspended in prewarmed DMEM +2% FCS at a concentration 106 /ml followed by a 1-hr incubation at 37°C with 4 µg/ml Hoechst 33342 (Sigma, St Louis, MO). ..

    Marker:

    Article Title: 18F-misonidazole PET imaging of hypoxia in micrometastases and macroscopic xenografts of human non-small cell lung cancer: a correlation with autoradiography and histological findings
    Article Snippet: The proliferation marker, bromodeoxyuridine (Roche Diagnostics, Indianapolis, IN) was first dissolved in dimethyl sulfoxide and further diluted in physiological saline to a final concentration of 20 mg/ml. .. The blood perfusion marker, Hoechst 33342 (Sigma-Aldrich, St. Louis, MO) was dissolved in physiological saline at a concentration of 5 mg/ml. .. In all cases, fresh drug solutions were prepared on the day of injection.

    Cell Cycle Assay:

    Article Title: Histone demethylase Lsd1 represses hematopoietic stem and progenitor cell signatures during blood cell maturation
    Article Snippet: Donor contribution in peripheral blood, mature bone marrow lineage, bone marrow stem cell and progenitor cell compartment, and spleen were measured by cell surface staining for the CD45.1 and CD45.2 markers as well as genomic DNA analysis of sorted populations. .. For cell cycle analysis, cells were resuspended in prewarmed DMEM +2% FCS at a concentration 106 /ml followed by a 1-hr incubation at 37°C with 4 µg/ml Hoechst 33342 (Sigma, St Louis, MO). ..

    Microscopy:

    Article Title: Myricetin induces apoptosis via endoplasmic reticulum stress and DNA double-strand breaks in human ovarian cancer cells
    Article Snippet: Immunofluorescent staining and confocal laser microscopy The cells were seeded onto coverslips in 24-well plates at a density of 5×104 cells/well 24 h prior to treatment. .. Following exposure to 40 µ g/ml myricetin for 0, 6, 12 and 24 h, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, stained with the nuclear stain Hoechst 33342 (2 µ g/ml; Sigma-Aldrich) for 2 min at room temperature, washed with PBS and examined using a confocal laser microscope (FV1000; Olympus, Tokyo, Japan) to reveal chromatin condensation. .. The expression levels of GRP-78, active Caspase 3 and γ-H2 AX were examined using an indirect immunofluorescence method.

    Imaging:

    Article Title: Development of an in vitro senescent hepatic cell model for metabolic studies in aging
    Article Snippet: Immunofluorescence imaging of intracellular lipids using BODIPY™ 493/503Control and senescent aml12 cells were seeded in 24-well plate at 7th day of the protocol, and cultured for 24 h. Cells were rinsed with 1x PBS and stained with BODIPY™ 493/503 (D3922; Molecular Probes, ThermoFisher Scientific) at 1:1000 dilution for 15 min. .. Cells were then rinsed with 1xPBS containing Hoechst 33342 (Sigma) to counter stain nucleus for 5 min. After rinse, cells were kept in 1x HBSS for imaging. .. Leica fluorescent microscope was used at 10x magnification for visualization and LAS X imaging software was used for image capture.

    other:

    Article Title: Drug-Selected Human Lung Cancer Stem Cells: Cytokine Network, Tumorigenic and Metastatic Properties
    Article Snippet: Reagents Cisplatin, doxorubicin, etoposide, and Hoechst 33342 were from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO).

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    Millipore hoechst 33342
    Apoptosis induction by resveratrol and labruscol in human skin melanoma cancer cells. Cells were treated during 48 h or 72 h with 50 μM resveratrol or labruscol. Apoptosis was assessed by staining (control cells or cells treated with resveratrol or labruscol) with 1 μg/mL Hoechst 33342. Co: control cells; RSV: resveratrol-treated cells; Labruscol: labruscol-treated cells. The values represented the means ± SD of at least 3 independent experiments. ***  p
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    Apoptosis induction by resveratrol and labruscol in human skin melanoma cancer cells. Cells were treated during 48 h or 72 h with 50 μM resveratrol or labruscol. Apoptosis was assessed by staining (control cells or cells treated with resveratrol or labruscol) with 1 μg/mL Hoechst 33342. Co: control cells; RSV: resveratrol-treated cells; Labruscol: labruscol-treated cells. The values represented the means ± SD of at least 3 independent experiments. ***  p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Cytotoxicity of Labruscol, a New Resveratrol Dimer Produced by Grapevine Cell Suspensions, on Human Skin Melanoma Cancer Cell Line HT-144

    doi: 10.3390/molecules22111940

    Figure Lengend Snippet: Apoptosis induction by resveratrol and labruscol in human skin melanoma cancer cells. Cells were treated during 48 h or 72 h with 50 μM resveratrol or labruscol. Apoptosis was assessed by staining (control cells or cells treated with resveratrol or labruscol) with 1 μg/mL Hoechst 33342. Co: control cells; RSV: resveratrol-treated cells; Labruscol: labruscol-treated cells. The values represented the means ± SD of at least 3 independent experiments. *** p

    Article Snippet: Invading cells on the lower side of the filter were stained with Hoechst 33342 (Sigma-Aldrich, Saint-Quentin, France).

    Techniques: Staining

    Apoptosis detection in p21+/+ and p21−/− podocytes in culture following treatment with ADR. A – D : representative images of podocytes stained with Hoechst 33342 following treatment with ADR ( A and C : p21+/+ podocytes; B and D : p21−/−

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: CDK inhibitor p21 is prosurvival in adriamycin-induced podocyte injury, in vitro and in vivo

    doi: 10.1152/ajprenal.00216.2009

    Figure Lengend Snippet: Apoptosis detection in p21+/+ and p21−/− podocytes in culture following treatment with ADR. A – D : representative images of podocytes stained with Hoechst 33342 following treatment with ADR ( A and C : p21+/+ podocytes; B and D : p21−/−

    Article Snippet: First, after growing podocytes to 80% confluence on plastic plates, apoptosis was measured by staining with Hoechst 33342 (Sigma, St. Louis, MO), as previously described ( ).

    Techniques: Staining

    Csf1r-iCre -targeted ECs proliferate in vivo. ( a,b ) E12.5 Csf1r-iCre;Rosa tdTom yolk sac ( a ) or hindbrain ( b ), wholemount stained for the proliferation marker pHH3 and VEGFR2 or for pHH3 together with IB4, respectively, and shown together with tdTom fluorescence (n = 3 each). Areas indicated with white squares were imaged at higher magnification and are shown below the corresponding panel, with tdTom and pHH3 channels also shown separately in grey scale. Symbols : The arrows indicate proliferating tdTom + pHH3 + ECs; solid and clear symbols indicate the presence or absence, respectively, of tdTom fluorescence; the wavy arrow indicates a tdTom - pHH3 + neural progenitor. Scale bars : 100 µm (top panels), 20 µm (lower panels). ( c-e ) Cell cycle distribution of tdTom + and tdTom - ECs . ( c ) FACS strategy to isolate tdTom + and tdTom - PECAM1 + ECs from E12.5 Csf1r-iCre;Rosa tdTom embryos (n = 3 embryos). ( d ) Graphic representation of cell cycle distribution based on Hoechst 33342 fluorescence as a measure of DNA content; low and high staining intensity is observed in cells with a DNA ploidy of 2n (G0/G1 phase) or 4n (G2/M phase), respectively; an intermediate staining intensity corresponds to S phase. ( e ) Mean ± SD proportion of tdTom + and tdTom - ECs in G1, S and G2/M based on the area of the corresponding peaks in ( d ); n.s., non-significant, P > 0.9999 (two-way ANOVA, Bonferroni’s multiple comparisons test).

    Journal: Nature

    Article Title: Erythro-myeloid progenitors contribute endothelial cells to blood vessels

    doi: 10.1038/s41586-018-0552-x

    Figure Lengend Snippet: Csf1r-iCre -targeted ECs proliferate in vivo. ( a,b ) E12.5 Csf1r-iCre;Rosa tdTom yolk sac ( a ) or hindbrain ( b ), wholemount stained for the proliferation marker pHH3 and VEGFR2 or for pHH3 together with IB4, respectively, and shown together with tdTom fluorescence (n = 3 each). Areas indicated with white squares were imaged at higher magnification and are shown below the corresponding panel, with tdTom and pHH3 channels also shown separately in grey scale. Symbols : The arrows indicate proliferating tdTom + pHH3 + ECs; solid and clear symbols indicate the presence or absence, respectively, of tdTom fluorescence; the wavy arrow indicates a tdTom - pHH3 + neural progenitor. Scale bars : 100 µm (top panels), 20 µm (lower panels). ( c-e ) Cell cycle distribution of tdTom + and tdTom - ECs . ( c ) FACS strategy to isolate tdTom + and tdTom - PECAM1 + ECs from E12.5 Csf1r-iCre;Rosa tdTom embryos (n = 3 embryos). ( d ) Graphic representation of cell cycle distribution based on Hoechst 33342 fluorescence as a measure of DNA content; low and high staining intensity is observed in cells with a DNA ploidy of 2n (G0/G1 phase) or 4n (G2/M phase), respectively; an intermediate staining intensity corresponds to S phase. ( e ) Mean ± SD proportion of tdTom + and tdTom - ECs in G1, S and G2/M based on the area of the corresponding peaks in ( d ); n.s., non-significant, P > 0.9999 (two-way ANOVA, Bonferroni’s multiple comparisons test).

    Article Snippet: For cell cycle analysis, cell populations were incubated with 10 µg/ml Hoechst 33342 (Sigma) for 30 mins at 37°C before labelling with PE/Cy7-conjugated rat anti-PECAM1 and performing FACS analysis.

    Techniques: In Vivo, Staining, Marker, Fluorescence, FACS

    The expression and combination with its noncompetitive antibody of 7D12. a The EGFR-antibody complex with one antibody and another noncompetitive single domain antibody on Hela cells. b SDS-PAGE analysis of 7D12 after being carried out in E. coli . In SDS-PAGE imaging, lane M was protein marker, lane 1 was protein expression by non-induced E. coli , lane 2 was protein expression by induced E. coli , lane 3 was supernatant after E. coli disruption, lane 4 was sedimentation after E. coli disruption. In the western blotting analysis of 7D12 after purification, lane M was protein marker, lane 1 was protein after purification. c 40 nM 7D12 (red) was treated on Hela cells for 1 h, cell nuclei were dyed by Hoechst 33342 (blue). Bars, 10 μm. d 40 nM 7D12 (red) were treated on Hela cells for 10 min, cell nuclei were dyed by Hoechst 33342 (blue). Bars, 10 μm. e 20 nM Cetuximab (green) and 20 nM 7D12 (red) were treated on Hela cells for 10 min, cell nuclei were dyed by Hoechst 33342 (blue). Bars, 10 μm

    Journal: Cancer Cell International

    Article Title: Lattice complex assembled by noncompetitive anti-EGFR antibodies regulates actin cytoskeletal reorganization

    doi: 10.1186/s12935-020-01204-z

    Figure Lengend Snippet: The expression and combination with its noncompetitive antibody of 7D12. a The EGFR-antibody complex with one antibody and another noncompetitive single domain antibody on Hela cells. b SDS-PAGE analysis of 7D12 after being carried out in E. coli . In SDS-PAGE imaging, lane M was protein marker, lane 1 was protein expression by non-induced E. coli , lane 2 was protein expression by induced E. coli , lane 3 was supernatant after E. coli disruption, lane 4 was sedimentation after E. coli disruption. In the western blotting analysis of 7D12 after purification, lane M was protein marker, lane 1 was protein after purification. c 40 nM 7D12 (red) was treated on Hela cells for 1 h, cell nuclei were dyed by Hoechst 33342 (blue). Bars, 10 μm. d 40 nM 7D12 (red) were treated on Hela cells for 10 min, cell nuclei were dyed by Hoechst 33342 (blue). Bars, 10 μm. e 20 nM Cetuximab (green) and 20 nM 7D12 (red) were treated on Hela cells for 10 min, cell nuclei were dyed by Hoechst 33342 (blue). Bars, 10 μm

    Article Snippet: Hoechst 33342 (Cat No.B2261), Chlorpromazine (Cat No.C0982), Nystatin (Cat No.N9150), Progesterone (Cat No.P0130) and Rapamycin (Cat No.V900930) were purchased from Sigma-Aldrich.

    Techniques: Expressing, SDS Page, Imaging, Marker, Sedimentation, Western Blot, Purification

    Lattice complex assembled along F-actin on cell membrane. a Different single antibody (40 nM) or combination of antibodies (20 nM each antibody) was treated on Hela cells for 10 min or 1 h, cell nuclei were dyed by Hoechst 33342 (blue). Afterward, F-actin was stained with AlexaFluor 488 Phalloidin. Bars, 10 μm. b 20 nM Cetuximab and 20 nM H11 (red) were treated on Hela cells for 10 min, F-actin was stained with AlexaFluor 488 Phalloidin. Arrowheads showed the lattice complex. Bars, 10 μm. c Surface rendering of images from ( b ) was generated from Imaris software. Bars, 1 μm. d , e Different combination of antibodies (20 nM each antibody) or single antibody (40 nM) was treated on Hela cells for 10 min, F-actin was stained with AlexaFluor 488 Phalloidin. Surface rendering of images was generated from Imaris software. Bars above, 10 μm, bars below, 10 μm

    Journal: Cancer Cell International

    Article Title: Lattice complex assembled by noncompetitive anti-EGFR antibodies regulates actin cytoskeletal reorganization

    doi: 10.1186/s12935-020-01204-z

    Figure Lengend Snippet: Lattice complex assembled along F-actin on cell membrane. a Different single antibody (40 nM) or combination of antibodies (20 nM each antibody) was treated on Hela cells for 10 min or 1 h, cell nuclei were dyed by Hoechst 33342 (blue). Afterward, F-actin was stained with AlexaFluor 488 Phalloidin. Bars, 10 μm. b 20 nM Cetuximab and 20 nM H11 (red) were treated on Hela cells for 10 min, F-actin was stained with AlexaFluor 488 Phalloidin. Arrowheads showed the lattice complex. Bars, 10 μm. c Surface rendering of images from ( b ) was generated from Imaris software. Bars, 1 μm. d , e Different combination of antibodies (20 nM each antibody) or single antibody (40 nM) was treated on Hela cells for 10 min, F-actin was stained with AlexaFluor 488 Phalloidin. Surface rendering of images was generated from Imaris software. Bars above, 10 μm, bars below, 10 μm

    Article Snippet: Hoechst 33342 (Cat No.B2261), Chlorpromazine (Cat No.C0982), Nystatin (Cat No.N9150), Progesterone (Cat No.P0130) and Rapamycin (Cat No.V900930) were purchased from Sigma-Aldrich.

    Techniques: Staining, Generated, Software

    Lattice complex assembled by EGFR and its different antibody combinations on cell membrane. a – c Different single antibody (40 nM) or combination of antibodies (20 nM each antibody) was treated on Hela cells for 1 h. Bars, 10 μm. d The lattice complex formed by EGFR and its noncompetitive antibodies on cell membrane when two EGFR antibodies with noncompetitive epitopes were treated on Hela cells. e The EGFR pattern with two competitive antibodies on Hela cells. f EGFR-antibody complex formed on cell membrane by single antibody. g, i Different single antibody (40 nM) or combination of antibodies (20 nM each antibody) was treated on Hela cells for 10 min, cell nuclei were dyed by Hoechst 33342 (blue). Arrowheads showed the lattice complex. Bars, 10 μm. j 20 nM Cetuximab (green) and 20 nM H11 (red) were treated on Hela cells for 10 min. Arrowheads showed the lattice complex. Surface rendering was generated from Imaris software. Bars in left 2 columns, 10 μm; in right 1 column, 1 μm

    Journal: Cancer Cell International

    Article Title: Lattice complex assembled by noncompetitive anti-EGFR antibodies regulates actin cytoskeletal reorganization

    doi: 10.1186/s12935-020-01204-z

    Figure Lengend Snippet: Lattice complex assembled by EGFR and its different antibody combinations on cell membrane. a – c Different single antibody (40 nM) or combination of antibodies (20 nM each antibody) was treated on Hela cells for 1 h. Bars, 10 μm. d The lattice complex formed by EGFR and its noncompetitive antibodies on cell membrane when two EGFR antibodies with noncompetitive epitopes were treated on Hela cells. e The EGFR pattern with two competitive antibodies on Hela cells. f EGFR-antibody complex formed on cell membrane by single antibody. g, i Different single antibody (40 nM) or combination of antibodies (20 nM each antibody) was treated on Hela cells for 10 min, cell nuclei were dyed by Hoechst 33342 (blue). Arrowheads showed the lattice complex. Bars, 10 μm. j 20 nM Cetuximab (green) and 20 nM H11 (red) were treated on Hela cells for 10 min. Arrowheads showed the lattice complex. Surface rendering was generated from Imaris software. Bars in left 2 columns, 10 μm; in right 1 column, 1 μm

    Article Snippet: Hoechst 33342 (Cat No.B2261), Chlorpromazine (Cat No.C0982), Nystatin (Cat No.N9150), Progesterone (Cat No.P0130) and Rapamycin (Cat No.V900930) were purchased from Sigma-Aldrich.

    Techniques: Generated, Software