fixed jurkat donor cells  (Millipore)


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    Millipore fixed jurkat donor cells
    Detection of rapid p8 transfer between <t>Jurkat</t> T-cells by flow cytometry. Jurkat donor T-cells were transfected with p8-HA or the control plasmid pME and co-cultured with prestained acceptor T-cells Jurkat-CMAC according to the experimental setup displayed in Figure 2 . (A) Flow cytometry. At 48 h post transfection, equal amounts of donor and acceptor cells (1 ∗ 10 6 cells each) were either directly fixed in 2% PFA and mixed (time point: 0 min), or they were co-cultured at <t>37°C</t> for 5, 30, 60 min, or 24 h before fixation. After intracellular staining using HA-specific, APC-labeled antibodies or the respective isotype-matched control antibodies, flow cytometry was performed. Representative dot plots at 60 min post co-culture are shown. First line: Dot plots display the forward scatter (FSC) plotted against the side scatter (SSC) and living cells are gated (red gate). Second line: CMAC-specific fluorescence is plotted against the SSC, which allows discrimination between CMAC-negative donor (purple gate) and CMAC-positive acceptor (blue gate) cells. Third line: HA-specific fluorescence is plotted against the SSC and numbers represent the efficiency of transfection ( E T ) within the CMAC-negative donor cells (displayed on the left) or the transfer of p8 ( T p8 ) within the CMAC-positive acceptor cells (displayed on the right). (B) Equation to calculate the relative transfer of p8 [ T p8(relative) ] between cells. T p8 shows the transfer of p8, which corresponds to the percentage of p8-HA positive cells within CMAC-positive acceptor cells (T p8(p8 t ) ) at a given time point t and which was normalized on background fluorescence of the respective control cells transfected with pME (T p8(pME t ) ). E T represents the efficiency of transfection at a given time point t and corresponds to the percentage of p8-HA positive cells within CMAC-negative donor cells (E T(p8 t ) ), which is corrected by background fluorescence of the respective control cells transfected with pME (E T(pME t ) ). (C) Time course analysis of T p8(relative) as measured by flow cytometry. The means of 3–4 independent experiments ±SE are shown and were compared as indicated using a paired t -test. ∗ Indicates p
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    Images

    1) Product Images from "Quantitating the Transfer of the HTLV-1 p8 Protein Between T-Cells by Flow Cytometry"

    Article Title: Quantitating the Transfer of the HTLV-1 p8 Protein Between T-Cells by Flow Cytometry

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00400

    Detection of rapid p8 transfer between Jurkat T-cells by flow cytometry. Jurkat donor T-cells were transfected with p8-HA or the control plasmid pME and co-cultured with prestained acceptor T-cells Jurkat-CMAC according to the experimental setup displayed in Figure 2 . (A) Flow cytometry. At 48 h post transfection, equal amounts of donor and acceptor cells (1 ∗ 10 6 cells each) were either directly fixed in 2% PFA and mixed (time point: 0 min), or they were co-cultured at 37°C for 5, 30, 60 min, or 24 h before fixation. After intracellular staining using HA-specific, APC-labeled antibodies or the respective isotype-matched control antibodies, flow cytometry was performed. Representative dot plots at 60 min post co-culture are shown. First line: Dot plots display the forward scatter (FSC) plotted against the side scatter (SSC) and living cells are gated (red gate). Second line: CMAC-specific fluorescence is plotted against the SSC, which allows discrimination between CMAC-negative donor (purple gate) and CMAC-positive acceptor (blue gate) cells. Third line: HA-specific fluorescence is plotted against the SSC and numbers represent the efficiency of transfection ( E T ) within the CMAC-negative donor cells (displayed on the left) or the transfer of p8 ( T p8 ) within the CMAC-positive acceptor cells (displayed on the right). (B) Equation to calculate the relative transfer of p8 [ T p8(relative) ] between cells. T p8 shows the transfer of p8, which corresponds to the percentage of p8-HA positive cells within CMAC-positive acceptor cells (T p8(p8 t ) ) at a given time point t and which was normalized on background fluorescence of the respective control cells transfected with pME (T p8(pME t ) ). E T represents the efficiency of transfection at a given time point t and corresponds to the percentage of p8-HA positive cells within CMAC-negative donor cells (E T(p8 t ) ), which is corrected by background fluorescence of the respective control cells transfected with pME (E T(pME t ) ). (C) Time course analysis of T p8(relative) as measured by flow cytometry. The means of 3–4 independent experiments ±SE are shown and were compared as indicated using a paired t -test. ∗ Indicates p
    Figure Legend Snippet: Detection of rapid p8 transfer between Jurkat T-cells by flow cytometry. Jurkat donor T-cells were transfected with p8-HA or the control plasmid pME and co-cultured with prestained acceptor T-cells Jurkat-CMAC according to the experimental setup displayed in Figure 2 . (A) Flow cytometry. At 48 h post transfection, equal amounts of donor and acceptor cells (1 ∗ 10 6 cells each) were either directly fixed in 2% PFA and mixed (time point: 0 min), or they were co-cultured at 37°C for 5, 30, 60 min, or 24 h before fixation. After intracellular staining using HA-specific, APC-labeled antibodies or the respective isotype-matched control antibodies, flow cytometry was performed. Representative dot plots at 60 min post co-culture are shown. First line: Dot plots display the forward scatter (FSC) plotted against the side scatter (SSC) and living cells are gated (red gate). Second line: CMAC-specific fluorescence is plotted against the SSC, which allows discrimination between CMAC-negative donor (purple gate) and CMAC-positive acceptor (blue gate) cells. Third line: HA-specific fluorescence is plotted against the SSC and numbers represent the efficiency of transfection ( E T ) within the CMAC-negative donor cells (displayed on the left) or the transfer of p8 ( T p8 ) within the CMAC-positive acceptor cells (displayed on the right). (B) Equation to calculate the relative transfer of p8 [ T p8(relative) ] between cells. T p8 shows the transfer of p8, which corresponds to the percentage of p8-HA positive cells within CMAC-positive acceptor cells (T p8(p8 t ) ) at a given time point t and which was normalized on background fluorescence of the respective control cells transfected with pME (T p8(pME t ) ). E T represents the efficiency of transfection at a given time point t and corresponds to the percentage of p8-HA positive cells within CMAC-negative donor cells (E T(p8 t ) ), which is corrected by background fluorescence of the respective control cells transfected with pME (E T(pME t ) ). (C) Time course analysis of T p8(relative) as measured by flow cytometry. The means of 3–4 independent experiments ±SE are shown and were compared as indicated using a paired t -test. ∗ Indicates p

    Techniques Used: Flow Cytometry, Cytometry, Transfection, Plasmid Preparation, Cell Culture, Staining, Labeling, Co-Culture Assay, Fluorescence

    Experimental setup of the co-culture assay. Jurkat T-cells were transfected with p8-HA expression plasmids or the control plasmid pME for 48 h. Transfected p8-donor or control (pME) cells were either subjected to immunoblot analysis or co-cultured with equal amounts of Jurkat acceptor cells (1 ∗ 10 6 ) that had been prestained with Cell Tracker Blue CMAC Dye (Jurkat-CMAC). At different time points post co-culture at 37°C (0, 5, 30, 60 min, 24 h), cells were fixed in 2% paraformaldehyde (PFA), permeabilized, stained and analyzed by flow cytometry.
    Figure Legend Snippet: Experimental setup of the co-culture assay. Jurkat T-cells were transfected with p8-HA expression plasmids or the control plasmid pME for 48 h. Transfected p8-donor or control (pME) cells were either subjected to immunoblot analysis or co-cultured with equal amounts of Jurkat acceptor cells (1 ∗ 10 6 ) that had been prestained with Cell Tracker Blue CMAC Dye (Jurkat-CMAC). At different time points post co-culture at 37°C (0, 5, 30, 60 min, 24 h), cells were fixed in 2% paraformaldehyde (PFA), permeabilized, stained and analyzed by flow cytometry.

    Techniques Used: Co-culture Assay, Transfection, Expressing, Plasmid Preparation, Cell Culture, Co-Culture Assay, Staining, Flow Cytometry, Cytometry

    Impairment of p8 transfer between T-cells after inhibition of actin polymerization. (A) Experimental setup. At 24 h post transfection of Jurkat T-cells with p8-HA or pME (control) expression plasmids (100 μg each), cells were cultured with increasing concentrations of an inhibitor of actin polymerization, cytochalasin D (0.5, 1, 2.5, 5 μM), or the solvent control dimethylsulfoxide (DMSO) for 24 h. Cells were either subjected to immunoblot analysis or co-cultured in fresh medium (without chemicals) with equal amounts of prestained Jurkat acceptor cells (1 ∗ 10 6 cells, labeled with Cell Tracker Blue CMAC Dye) for 24 h at 37°C and analyzed by flow cytometry. DMSO-treated donor cells were also taken at 0 h post co-culture and served as negative control for p8 transfer. (B) The relative transfer of p8 [ T p8(relative) ] was calculated as explained in Figure 3 . Values display the means of at least four independent experiments (±SE) and were normalized on and compared to those of cells treated with DMSO and co-cultured for 24 h using an unpaired t -test. ∗∗ Indicates p
    Figure Legend Snippet: Impairment of p8 transfer between T-cells after inhibition of actin polymerization. (A) Experimental setup. At 24 h post transfection of Jurkat T-cells with p8-HA or pME (control) expression plasmids (100 μg each), cells were cultured with increasing concentrations of an inhibitor of actin polymerization, cytochalasin D (0.5, 1, 2.5, 5 μM), or the solvent control dimethylsulfoxide (DMSO) for 24 h. Cells were either subjected to immunoblot analysis or co-cultured in fresh medium (without chemicals) with equal amounts of prestained Jurkat acceptor cells (1 ∗ 10 6 cells, labeled with Cell Tracker Blue CMAC Dye) for 24 h at 37°C and analyzed by flow cytometry. DMSO-treated donor cells were also taken at 0 h post co-culture and served as negative control for p8 transfer. (B) The relative transfer of p8 [ T p8(relative) ] was calculated as explained in Figure 3 . Values display the means of at least four independent experiments (±SE) and were normalized on and compared to those of cells treated with DMSO and co-cultured for 24 h using an unpaired t -test. ∗∗ Indicates p

    Techniques Used: Inhibition, Transfection, Expressing, Cell Culture, Labeling, Flow Cytometry, Cytometry, Co-Culture Assay, Negative Control

    Detection of p8 transfer between Jurkat T-cells by immunofluorescence. (A) Jurkat T-cells were transfected with expression plasmids p8-HA or pME for 48 h and co-cultivated with equal amounts of acceptor Jurkat T-cells prestained with Cell Tracker Blue CMAC (Jurkat-CMAC) on poly- L -lysine coated glass slides for 24 h at 37°C. Thereafter, cells were permeabilized and stained with HA-specific antibodies and the respective secondary antibodies. Slides were covered with ProLong Gold antifade reagent and analyzed by confocal microscopy. A cutout of an optical field shows cells expressing p8-HA (red) within the donor Jurkat T-cells (not stained) and the acceptor Jurkat T-cells (blue). The numbers of p8-positive cells (red) within the acceptor Jurkat T-cells (blue) were counted (white circles). Red arrows: p8-positive donor cells; blue arrows: p8-positive acceptor cell; blow up: example of a p8-expressing acceptor cell; white arrows: p8-HA. (B) Comparison of p8 transfer between flow cytometry (black bars) and immunofluorescence (gray bars). At 48 h post transfection with p8-HA, equal amounts of p8-donor Jurkat T- cells and Jurkat-CMAC acceptor cells (1 ∗ 10 6 cells each) were co-cultured at 37°C for 5, 30, 60 min or 24 h before fixation. One representative time course experiment of relative p8 transfer [ T p8(relative) ] as measured by flow cytometry (as shown for n = 4 in Figure 3C ) is compared to the manual quantitation of relative p8 transfer within the same sample by immunofluorescence. T p8(relative) as measured by immunofluorescence was calculated by normalizing the mean percentage of p8-HA positive cells within CMAC-positive acceptor cells on the mean percentage of p8-HA positive cells within CMAC-negative donor cells in 20 optical fields. SE, standard error.
    Figure Legend Snippet: Detection of p8 transfer between Jurkat T-cells by immunofluorescence. (A) Jurkat T-cells were transfected with expression plasmids p8-HA or pME for 48 h and co-cultivated with equal amounts of acceptor Jurkat T-cells prestained with Cell Tracker Blue CMAC (Jurkat-CMAC) on poly- L -lysine coated glass slides for 24 h at 37°C. Thereafter, cells were permeabilized and stained with HA-specific antibodies and the respective secondary antibodies. Slides were covered with ProLong Gold antifade reagent and analyzed by confocal microscopy. A cutout of an optical field shows cells expressing p8-HA (red) within the donor Jurkat T-cells (not stained) and the acceptor Jurkat T-cells (blue). The numbers of p8-positive cells (red) within the acceptor Jurkat T-cells (blue) were counted (white circles). Red arrows: p8-positive donor cells; blue arrows: p8-positive acceptor cell; blow up: example of a p8-expressing acceptor cell; white arrows: p8-HA. (B) Comparison of p8 transfer between flow cytometry (black bars) and immunofluorescence (gray bars). At 48 h post transfection with p8-HA, equal amounts of p8-donor Jurkat T- cells and Jurkat-CMAC acceptor cells (1 ∗ 10 6 cells each) were co-cultured at 37°C for 5, 30, 60 min or 24 h before fixation. One representative time course experiment of relative p8 transfer [ T p8(relative) ] as measured by flow cytometry (as shown for n = 4 in Figure 3C ) is compared to the manual quantitation of relative p8 transfer within the same sample by immunofluorescence. T p8(relative) as measured by immunofluorescence was calculated by normalizing the mean percentage of p8-HA positive cells within CMAC-positive acceptor cells on the mean percentage of p8-HA positive cells within CMAC-negative donor cells in 20 optical fields. SE, standard error.

    Techniques Used: Immunofluorescence, Transfection, Expressing, Staining, Confocal Microscopy, Flow Cytometry, Cytometry, Cell Culture, Quantitation Assay

    Cell type-dependence of an efficient p8 transfer. (A,B) 293T were transfected with p8-HA expression plasmids or the control plasmid pME for 48h. Transfected p8-donor or control (pME) cells were co-cultured with equal amounts of Jurkat acceptor cells (3 ∗ 10 6 ) that had been prestained with Cell Tracker Blue CMAC Dye. At 5 min or at 24 h post co-culture at 37°C, cells were fixed in 2% PFA, permeabilized, stained and analyzed by flow cytometry. (A) Representative dot plots of 293T cells transfected with p8-HA expression plasmids after co-culture with Jurkat-CMAC acceptor cells for 24 h are shown. First line: Dot plots display the forward scatter (FSC) plotted against the side scatter (SSC) and living cells are gated (red gate). Second line: CMAC-specific fluorescence is plotted against the SSC, which allows discrimination between CMAC-negative 293T donor (purple gate) and CMAC-positive Jurkat acceptor (blue gate) cells. Third line: HA-specific fluorescence is plotted against the SSC and numbers represent the efficiency of transfection (E T ) within the CMAC-negative 293T donor cells (displayed on the left) or the transfer of p8 ( T p8 ) within the CMAC-positive Jurkat acceptor cells (displayed on the right). (B) Time course analysis of T p8(relative) as measured by flow cytometry in co-cultures of 293T and Jurkat T-cells. The means of 4 independent experiments ±SE are shown and were compared as indicated using a t -test. For comparison, T p8(relative) between co-cultured Jurkat T-cells as shown in Figure 3C is displayed. (C) Jurkat donor T-cells were transfected with p8-HA or the control plasmid pME. At 48 h post transfection, 1 ∗ 10 6 donor cells were either fixed on poly- L -lysine coated culture plates for 1 h or they were left untreated. Thereafter, Jurkat donor T-cells were co-cultured with equal amounts of prestained acceptor T-cells Jurkat-CMAC at 37°C for 24 h and T p8(relative) was analyzed by flow cytometry. The means of 3 independent experiments ±SE are shown and were compared as indicated using an unpaired t -test. ∗∗ Indicates p
    Figure Legend Snippet: Cell type-dependence of an efficient p8 transfer. (A,B) 293T were transfected with p8-HA expression plasmids or the control plasmid pME for 48h. Transfected p8-donor or control (pME) cells were co-cultured with equal amounts of Jurkat acceptor cells (3 ∗ 10 6 ) that had been prestained with Cell Tracker Blue CMAC Dye. At 5 min or at 24 h post co-culture at 37°C, cells were fixed in 2% PFA, permeabilized, stained and analyzed by flow cytometry. (A) Representative dot plots of 293T cells transfected with p8-HA expression plasmids after co-culture with Jurkat-CMAC acceptor cells for 24 h are shown. First line: Dot plots display the forward scatter (FSC) plotted against the side scatter (SSC) and living cells are gated (red gate). Second line: CMAC-specific fluorescence is plotted against the SSC, which allows discrimination between CMAC-negative 293T donor (purple gate) and CMAC-positive Jurkat acceptor (blue gate) cells. Third line: HA-specific fluorescence is plotted against the SSC and numbers represent the efficiency of transfection (E T ) within the CMAC-negative 293T donor cells (displayed on the left) or the transfer of p8 ( T p8 ) within the CMAC-positive Jurkat acceptor cells (displayed on the right). (B) Time course analysis of T p8(relative) as measured by flow cytometry in co-cultures of 293T and Jurkat T-cells. The means of 4 independent experiments ±SE are shown and were compared as indicated using a t -test. For comparison, T p8(relative) between co-cultured Jurkat T-cells as shown in Figure 3C is displayed. (C) Jurkat donor T-cells were transfected with p8-HA or the control plasmid pME. At 48 h post transfection, 1 ∗ 10 6 donor cells were either fixed on poly- L -lysine coated culture plates for 1 h or they were left untreated. Thereafter, Jurkat donor T-cells were co-cultured with equal amounts of prestained acceptor T-cells Jurkat-CMAC at 37°C for 24 h and T p8(relative) was analyzed by flow cytometry. The means of 3 independent experiments ±SE are shown and were compared as indicated using an unpaired t -test. ∗∗ Indicates p

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Cell Culture, Co-Culture Assay, Staining, Flow Cytometry, Cytometry, Fluorescence

    2) Product Images from "IgG-mediated immune suppression in mice is epitope specific except during high epitope density conditions"

    Article Title: IgG-mediated immune suppression in mice is epitope specific except during high epitope density conditions

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33087-6

    IgG anti-NP suppresses IgM anti-SRBC responses at high, but not at low, epitope density. SRBC-specific IgM responses were analyzed in the mice described in Fig. 2 . ( A ) SRBC-specific IgM-secreting cells per spleen (=Exp 1 in B and C ). ( B , C ) Summary of the PFC results from all four experiments performed. Suppression is presented as the percentage of the control responses (number of PFCs in mice immunized with SRBC-NP alone, 100%, dashed line) that remains in mice immunized with IgG anti-NP+ respective SRBC-NP. The mean of PFCs in the control groups (representing 100%) were, in the order SRBC-NP high/int/low : 30, 080/71, 300/52, 533 (Exp. 1); 24, 793/50, 960/39, 200 (Exp 2); 30, 267/73, 233/136, 027 (Exp. 3); 22, 308/33, 267/63, 967 (Exp 4). p values represent comparisons between groups that received IgG anti-NP + SRBC-NP high/int/low and only SRBC-NP high/int/low . Each experiment is shown with a different symbol. ( D ) Serum IgM anti-SRBC levels (serum dilution in ELISA = 1:625). The dashed line indicates the mean value of mice immunized with IgG anti-NP alone. ns = p > 0.05, * p
    Figure Legend Snippet: IgG anti-NP suppresses IgM anti-SRBC responses at high, but not at low, epitope density. SRBC-specific IgM responses were analyzed in the mice described in Fig. 2 . ( A ) SRBC-specific IgM-secreting cells per spleen (=Exp 1 in B and C ). ( B , C ) Summary of the PFC results from all four experiments performed. Suppression is presented as the percentage of the control responses (number of PFCs in mice immunized with SRBC-NP alone, 100%, dashed line) that remains in mice immunized with IgG anti-NP+ respective SRBC-NP. The mean of PFCs in the control groups (representing 100%) were, in the order SRBC-NP high/int/low : 30, 080/71, 300/52, 533 (Exp. 1); 24, 793/50, 960/39, 200 (Exp 2); 30, 267/73, 233/136, 027 (Exp. 3); 22, 308/33, 267/63, 967 (Exp 4). p values represent comparisons between groups that received IgG anti-NP + SRBC-NP high/int/low and only SRBC-NP high/int/low . Each experiment is shown with a different symbol. ( D ) Serum IgM anti-SRBC levels (serum dilution in ELISA = 1:625). The dashed line indicates the mean value of mice immunized with IgG anti-NP alone. ns = p > 0.05, * p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    IgG anti-NP suppresses IgG anti-NP but not IgG anti-SRBC responses at all NP densities. C57BL/6 mice (5 per group) were immunized in the same way as described in Fig. 2 . Serum samples were collected 7, 21, and 35 days after immunization and assayed for NP- and SRBC-specific IgG b levels (serum dilution in ELISA = 1:625). p values represent comparisons between groups that received IgG anti-NP + SRBC-NP high/int/low and only SRBC-NP high/int/low . ns = p > 0.05, * p
    Figure Legend Snippet: IgG anti-NP suppresses IgG anti-NP but not IgG anti-SRBC responses at all NP densities. C57BL/6 mice (5 per group) were immunized in the same way as described in Fig. 2 . Serum samples were collected 7, 21, and 35 days after immunization and assayed for NP- and SRBC-specific IgG b levels (serum dilution in ELISA = 1:625). p values represent comparisons between groups that received IgG anti-NP + SRBC-NP high/int/low and only SRBC-NP high/int/low . ns = p > 0.05, * p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    IgG anti-NP suppresses IgM anti-NP responses at all NP densities. C57BL/6 mice were immunized i.v. with 5 × 10 7 SRBC-NP with high (SRBC-NP high ), intermediate (SRBC-NP int ) or low (SRBC-NP low ) epitope density with or without 30 μg polyclonal IgG anti-NP. Mice immunized with 5 × 10 7 unconjugated SRBC or with 30 μg IgG-anti NP only were used as controls. NP-specific immune responses were analyzed in spleen and serum samples obtained 5 days after immunization. ( A ) Cells were initially gated for B220 + cells. Representative flow cytometry gating for B220 + GC (defined as GL7 high CD95 high ) and non-GC λ1 + NP + cells from mice immunized with SRBC-NP high (left), IgG anti-NP + SRBC-NP high (middle) and IgG anti-NP alone (right). ( B ) Frequency of GC and non-GC NP + λ1 + cells in total B220 + cells. ( C ) NP-specific IgM-secreting cells per spleen. ( D ) Serum IgM anti-NP levels (serum dilution in ELISA = 1:625). The dashed line indicates the mean value of mice immunized with unconjugated SRBC. Representative of four independent experiments with 4–5 mice per group. ns = p > 0.05, * p
    Figure Legend Snippet: IgG anti-NP suppresses IgM anti-NP responses at all NP densities. C57BL/6 mice were immunized i.v. with 5 × 10 7 SRBC-NP with high (SRBC-NP high ), intermediate (SRBC-NP int ) or low (SRBC-NP low ) epitope density with or without 30 μg polyclonal IgG anti-NP. Mice immunized with 5 × 10 7 unconjugated SRBC or with 30 μg IgG-anti NP only were used as controls. NP-specific immune responses were analyzed in spleen and serum samples obtained 5 days after immunization. ( A ) Cells were initially gated for B220 + cells. Representative flow cytometry gating for B220 + GC (defined as GL7 high CD95 high ) and non-GC λ1 + NP + cells from mice immunized with SRBC-NP high (left), IgG anti-NP + SRBC-NP high (middle) and IgG anti-NP alone (right). ( B ) Frequency of GC and non-GC NP + λ1 + cells in total B220 + cells. ( C ) NP-specific IgM-secreting cells per spleen. ( D ) Serum IgM anti-NP levels (serum dilution in ELISA = 1:625). The dashed line indicates the mean value of mice immunized with unconjugated SRBC. Representative of four independent experiments with 4–5 mice per group. ns = p > 0.05, * p

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    3) Product Images from "Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells"

    Article Title: Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02852

    Ndr2-deficiency in murine CD4 + T cells attenuates TCR-induced FLNa phosphorylation at S2152, T-cell adhesion and LFA-1-dependent upregulation of CD69 in vitro . (A) Purified splenic wild type (WT) and Ndr2 −/− CD4 + T cells were left untreated or stimulated with anti-CD3 antibodies for the indicated time points. Lysates were prepared and analyzed by Western blotting using the indicated antibodies. Densitrometric quantification of FLNa phosphorylation at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 3). (B) Purified splenic WT and Ndr2 −/− CD4 + T cells were left untreated (non) or stimulated with anti-CD3 antibodies (CD3) and subsequently analyzed for their ability to bind plate-bound Fc-ICAM-1. Adherent cells were counted and calculated as percentage of input ( n = 3). (C) Purified splenic CD4 + T cells from WT and Ndr2 mice were cultured with plate-bound anti-CD3 antibodies (CD3) in the absence or presence of Fc-ICAM-1 (ICAM-1) with or without blocking LFA-1 antibodies (LFA-1) for 12 h. The upregulation of the activation marker CD69 of unstimulated (0 h) or activated T cells (12 h) were assessed by flow cytometry to determine the mean fluorescence intensity (MFI) ( n = 3). (mean ± SEM; * p ≤ 0.05, ** p ≤ 0.01).
    Figure Legend Snippet: Ndr2-deficiency in murine CD4 + T cells attenuates TCR-induced FLNa phosphorylation at S2152, T-cell adhesion and LFA-1-dependent upregulation of CD69 in vitro . (A) Purified splenic wild type (WT) and Ndr2 −/− CD4 + T cells were left untreated or stimulated with anti-CD3 antibodies for the indicated time points. Lysates were prepared and analyzed by Western blotting using the indicated antibodies. Densitrometric quantification of FLNa phosphorylation at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 3). (B) Purified splenic WT and Ndr2 −/− CD4 + T cells were left untreated (non) or stimulated with anti-CD3 antibodies (CD3) and subsequently analyzed for their ability to bind plate-bound Fc-ICAM-1. Adherent cells were counted and calculated as percentage of input ( n = 3). (C) Purified splenic CD4 + T cells from WT and Ndr2 mice were cultured with plate-bound anti-CD3 antibodies (CD3) in the absence or presence of Fc-ICAM-1 (ICAM-1) with or without blocking LFA-1 antibodies (LFA-1) for 12 h. The upregulation of the activation marker CD69 of unstimulated (0 h) or activated T cells (12 h) were assessed by flow cytometry to determine the mean fluorescence intensity (MFI) ( n = 3). (mean ± SEM; * p ≤ 0.05, ** p ≤ 0.01).

    Techniques Used: In Vitro, Purification, Western Blot, Mouse Assay, Cell Culture, Blocking Assay, Activation Assay, Marker, Flow Cytometry, Cytometry, Fluorescence

    Activated Ndr2 releases FLNa binding from LFA-1. (A) Jurkat T cells were transfected with constructs that suppress endogenous Ndr2 (shNdr2) and re-express a FLAG-tagged shRNA-resistant wild type (WT Ndr2) or a kinase-dead mutant of Ndr2 (K119A Ndr2). 48 h after transfection, whole-cell extracts were prepared and analyzed by Western blotting using the indicated antibodies. Numbers represent the reduction and re-expression of Ndr2 and its mutant after normalization to the Ndr2 expression level of shC-tranfected control cells. (B,C) Cells left untreated or stimulated for the indicated time points with CD3 antibodies. Lysates were used for immunoprecipitation of LFA-1 using anti-CD11a antibodies. Precipitates were divided and analyzed by Western blotting for FLNa, Talin and Kindlin-3 association. Densitrometric analyses of FLNa, Talin, or Kindlin-3 associated to LFA-1 are depicted in Figure S8 .
    Figure Legend Snippet: Activated Ndr2 releases FLNa binding from LFA-1. (A) Jurkat T cells were transfected with constructs that suppress endogenous Ndr2 (shNdr2) and re-express a FLAG-tagged shRNA-resistant wild type (WT Ndr2) or a kinase-dead mutant of Ndr2 (K119A Ndr2). 48 h after transfection, whole-cell extracts were prepared and analyzed by Western blotting using the indicated antibodies. Numbers represent the reduction and re-expression of Ndr2 and its mutant after normalization to the Ndr2 expression level of shC-tranfected control cells. (B,C) Cells left untreated or stimulated for the indicated time points with CD3 antibodies. Lysates were used for immunoprecipitation of LFA-1 using anti-CD11a antibodies. Precipitates were divided and analyzed by Western blotting for FLNa, Talin and Kindlin-3 association. Densitrometric analyses of FLNa, Talin, or Kindlin-3 associated to LFA-1 are depicted in Figure S8 .

    Techniques Used: Binding Assay, Transfection, Construct, shRNA, Mutagenesis, Western Blot, Expressing, Immunoprecipitation

    Kinase activity of Ndr2 controls TCR-mediated adhesion, interaction of T cells with APCs and LFA-1 activation. (A) Schematic representation of the suppression/re-expression plasmids for Ndr2 used in this study. (B) Jurkat T cells were transfected with suppression/re-expression plasmids which do not suppress endogenous Ndr2 (shC), reduce the endogenous protein level of Ndr2 (shNdr2), re-express a FLAG-tagged shRNA-resistant wild type Ndr2 (WT Ndr2) or re-express its kinase dead mutant (K119A Ndr2). 48 h after transfection, lysates were analyzed by Western blotting for Ndr2, Ndr1, FLAG, and β-actin (loading control). Numbers represent the reduction and re-expression of Ndr2 and its mutant after normalization to the Ndr2 expression level of the shC-tranfected control cells, which were set to 1 ( n = 4; right graph). (C) Transfected Jurkat T cells as described in (B) were analyzed for their ability to adhere to ICAM-1-coated wells in a resting state or stimulated for 30 min with CD3 antibodies. Adherent cells were counted and calculated as percentage of input ( n = 4). (D) Cells were transfected as described in (B) and analyzed for their ability to form conjugates with DDAO-SE (red)-stained Raji B cells that were pulsed without (non) or with superantigen (SA) for 30 min. The percentage of conjugates was defined as the number of double positive events in the upper right quadrant ( n = 4). (E) Jurkat T cells transfected as described in (B) were left untreated (non) or stimulated with CD3 antibodies (CD3), followed by staining with the anti-LFA-1 antibody mAb24 which recognizes the high affinity conformation of LFA-1. mAb24 epitope expression was assessed by flow cytometry within the GFP gate and data are normalized against LFA-1 expression detected by MEM48 ( n = 4). (mean ± SEM; * p ≤ 0.05; *** p ≤ 0.001).
    Figure Legend Snippet: Kinase activity of Ndr2 controls TCR-mediated adhesion, interaction of T cells with APCs and LFA-1 activation. (A) Schematic representation of the suppression/re-expression plasmids for Ndr2 used in this study. (B) Jurkat T cells were transfected with suppression/re-expression plasmids which do not suppress endogenous Ndr2 (shC), reduce the endogenous protein level of Ndr2 (shNdr2), re-express a FLAG-tagged shRNA-resistant wild type Ndr2 (WT Ndr2) or re-express its kinase dead mutant (K119A Ndr2). 48 h after transfection, lysates were analyzed by Western blotting for Ndr2, Ndr1, FLAG, and β-actin (loading control). Numbers represent the reduction and re-expression of Ndr2 and its mutant after normalization to the Ndr2 expression level of the shC-tranfected control cells, which were set to 1 ( n = 4; right graph). (C) Transfected Jurkat T cells as described in (B) were analyzed for their ability to adhere to ICAM-1-coated wells in a resting state or stimulated for 30 min with CD3 antibodies. Adherent cells were counted and calculated as percentage of input ( n = 4). (D) Cells were transfected as described in (B) and analyzed for their ability to form conjugates with DDAO-SE (red)-stained Raji B cells that were pulsed without (non) or with superantigen (SA) for 30 min. The percentage of conjugates was defined as the number of double positive events in the upper right quadrant ( n = 4). (E) Jurkat T cells transfected as described in (B) were left untreated (non) or stimulated with CD3 antibodies (CD3), followed by staining with the anti-LFA-1 antibody mAb24 which recognizes the high affinity conformation of LFA-1. mAb24 epitope expression was assessed by flow cytometry within the GFP gate and data are normalized against LFA-1 expression detected by MEM48 ( n = 4). (mean ± SEM; * p ≤ 0.05; *** p ≤ 0.001).

    Techniques Used: Activity Assay, Activation Assay, Expressing, Transfection, shRNA, Mutagenesis, Western Blot, Staining, Flow Cytometry, Cytometry

    Ndr2 phosphorylates FLNa at S2152 in vitro . (A) Purified WT Ndr2/Mob2 heterodimer was used to phosphorylate a positional scanning peptide library using radiolabeled ATP. The degree of phosphorylation of each component of the library, harboring the indicated amino acid residue at the indicated position relative to the phosphorylation site, is shown at left. Quantified data were normalized, log 2 transformed, and used to generate a heat map shown at right ( n = 2). (B) HEK 293T cells were transfected with either empty pEFBOS vector (vector) or plasmids encoding FLAG-tagged wild type Ndr2 (FNdr2) and a kinase dead (K119A) mutant of Ndr2 (FNdr2K119A). Cells were left untreated or treated with okadaic acid (OA), lysed and Ndr2 was immunoprecipitated using FLAG antibodies. A GST-FLNa fragment (19–24 repeats) was used as substrate for an in vitro kinase assay. Reactions were analyzed by Western blotting with the indicated antibodies ( n = 3). (C) Jurkat T cells were left untreated or stimulated for the indicated time points with CD3 antibodies. Cells were lysed and analyzed by Western Blotting with the indicated antibodies. Aliquots of whole-cell extracts were analyzed for the phosphorylation status of ERK1/2 to verify successful stimulation of T cells. Densitrometric analysis of the FLNa phosphorylation status at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 4) (mean ± SEM; ** p ≤ 0.01).
    Figure Legend Snippet: Ndr2 phosphorylates FLNa at S2152 in vitro . (A) Purified WT Ndr2/Mob2 heterodimer was used to phosphorylate a positional scanning peptide library using radiolabeled ATP. The degree of phosphorylation of each component of the library, harboring the indicated amino acid residue at the indicated position relative to the phosphorylation site, is shown at left. Quantified data were normalized, log 2 transformed, and used to generate a heat map shown at right ( n = 2). (B) HEK 293T cells were transfected with either empty pEFBOS vector (vector) or plasmids encoding FLAG-tagged wild type Ndr2 (FNdr2) and a kinase dead (K119A) mutant of Ndr2 (FNdr2K119A). Cells were left untreated or treated with okadaic acid (OA), lysed and Ndr2 was immunoprecipitated using FLAG antibodies. A GST-FLNa fragment (19–24 repeats) was used as substrate for an in vitro kinase assay. Reactions were analyzed by Western blotting with the indicated antibodies ( n = 3). (C) Jurkat T cells were left untreated or stimulated for the indicated time points with CD3 antibodies. Cells were lysed and analyzed by Western Blotting with the indicated antibodies. Aliquots of whole-cell extracts were analyzed for the phosphorylation status of ERK1/2 to verify successful stimulation of T cells. Densitrometric analysis of the FLNa phosphorylation status at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 4) (mean ± SEM; ** p ≤ 0.01).

    Techniques Used: In Vitro, Purification, Transformation Assay, Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Kinase Assay, Western Blot

    Ndr2 phosphorylates FLNa at S2152 in Jurkat T cells in vivo . (A) Jurkat T cells were transfected with suppression/re-expression constructs which suppress endogenous Ndr2 (shNdr2) and re-express a FLAG-tagged shRNA-resistant wild type (WT Ndr2) or a kinase-dead mutant of Ndr2 (K119A Ndr2). Numbers represent the reduction and re-expression of Ndr2 and its mutant after normalization to the Ndr2 expression level of the shC-tranfected control cells. (B) At 48 h post-transfection, cells left untreated or stimulated for the indicated time points with CD3 antibodies. Cells were lysed and analyzed by Western Blotting with the indicated antibodies. Densitrometric quantification of FLNa phosphorylation at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 3). (mean ± SEM; ** p ≤ 0.05).
    Figure Legend Snippet: Ndr2 phosphorylates FLNa at S2152 in Jurkat T cells in vivo . (A) Jurkat T cells were transfected with suppression/re-expression constructs which suppress endogenous Ndr2 (shNdr2) and re-express a FLAG-tagged shRNA-resistant wild type (WT Ndr2) or a kinase-dead mutant of Ndr2 (K119A Ndr2). Numbers represent the reduction and re-expression of Ndr2 and its mutant after normalization to the Ndr2 expression level of the shC-tranfected control cells. (B) At 48 h post-transfection, cells left untreated or stimulated for the indicated time points with CD3 antibodies. Cells were lysed and analyzed by Western Blotting with the indicated antibodies. Densitrometric quantification of FLNa phosphorylation at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 3). (mean ± SEM; ** p ≤ 0.05).

    Techniques Used: In Vivo, Transfection, Expressing, Construct, shRNA, Mutagenesis, Western Blot

    Expression profile, activation status and localization of Ndr2 in primary lymphocytes and lymphocyte-derived cell lines. (A) Total cell lysates of primary human and murine lymphocytes, Jurkat T cells, Raji B cells and HEK 293T cells were analyzed by Western blotting for expression of Ndr2 and Ndr1. β-actin staining served as loading control. Densitrometric analysis was performed to determine the Ndr2/Ndr1 ratio ( n = 3; right graph). (B) Jurkat T cells were stimulated with CD3 antibodies for the indicated time points. Cells were lysed and Ndr2 was immunoprecipitated using Ndr2 rabbit antibody. Ndr2-precipitates were divided and one half of the precipitates was used to assess Ndr2 kinase activity by an in vitro kinase assay (IVK) using the myelin basic protein (MBP) as substrate. Phosphorylation of MBP was visualized with autoradiography. Densitrometric analysis were performed to determine the intensity of all MBP bands and values of MBP intensities from time point 0 min were set to 1 ( n = 2; right graph). The second half of precipitates was used to detect Ndr2 by Western blotting. Aliquots of whole-cell extracts were analyzed for the phosphorylation status of ERK1/2 to verify successful stimulation of T cells (Input/lower panel). (C) Splenic B cells were loaded with OVA-peptide and co-incubated with purified T cells derived from OVA-TCR transgenic DO11.10 mice for 30 min. Cells were fixed, permeabilized and stained with an anti-Ndr2 Abs in combination with anti-rabbit IgG-FITC (green). F-actin was visualized with TRITC-Phalloidin (red) (upper panel). T/B cell conjugates were stained with Cy3-labeled anti-CD3 mAbs (red) and for Ndr2 (green; as described above; lower panel). Cells were imaged by confocal microscopy. Representative conjugates are shown. Each study was repeated at least three times and more than 25 conjugates were examined per condition. Scale bars define 5 μm. (mean ± SEM).
    Figure Legend Snippet: Expression profile, activation status and localization of Ndr2 in primary lymphocytes and lymphocyte-derived cell lines. (A) Total cell lysates of primary human and murine lymphocytes, Jurkat T cells, Raji B cells and HEK 293T cells were analyzed by Western blotting for expression of Ndr2 and Ndr1. β-actin staining served as loading control. Densitrometric analysis was performed to determine the Ndr2/Ndr1 ratio ( n = 3; right graph). (B) Jurkat T cells were stimulated with CD3 antibodies for the indicated time points. Cells were lysed and Ndr2 was immunoprecipitated using Ndr2 rabbit antibody. Ndr2-precipitates were divided and one half of the precipitates was used to assess Ndr2 kinase activity by an in vitro kinase assay (IVK) using the myelin basic protein (MBP) as substrate. Phosphorylation of MBP was visualized with autoradiography. Densitrometric analysis were performed to determine the intensity of all MBP bands and values of MBP intensities from time point 0 min were set to 1 ( n = 2; right graph). The second half of precipitates was used to detect Ndr2 by Western blotting. Aliquots of whole-cell extracts were analyzed for the phosphorylation status of ERK1/2 to verify successful stimulation of T cells (Input/lower panel). (C) Splenic B cells were loaded with OVA-peptide and co-incubated with purified T cells derived from OVA-TCR transgenic DO11.10 mice for 30 min. Cells were fixed, permeabilized and stained with an anti-Ndr2 Abs in combination with anti-rabbit IgG-FITC (green). F-actin was visualized with TRITC-Phalloidin (red) (upper panel). T/B cell conjugates were stained with Cy3-labeled anti-CD3 mAbs (red) and for Ndr2 (green; as described above; lower panel). Cells were imaged by confocal microscopy. Representative conjugates are shown. Each study was repeated at least three times and more than 25 conjugates were examined per condition. Scale bars define 5 μm. (mean ± SEM).

    Techniques Used: Expressing, Activation Assay, Derivative Assay, Western Blot, Staining, Immunoprecipitation, Activity Assay, In Vitro, Kinase Assay, Autoradiography, Incubation, Purification, Transgenic Assay, Mouse Assay, Labeling, Confocal Microscopy

    4) Product Images from "Human Mast Cells From Adipose Tissue Target and Induce Apoptosis of Breast Cancer Cells"

    Article Title: Human Mast Cells From Adipose Tissue Target and Induce Apoptosis of Breast Cancer Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00138

    ADMC killing of human breast cancer cells measured as evidence by the uptake of PI. (A) CellTracker™-red labeled ADMC (7.5 × 10 4 ; shown here as purple cells) were sensitized with 1 μg/ml anti-HER2/ neu IgE (clone trastuzumab), washed, and incubated with MitoTracker™-green-stained SK-BR-3 (10 5 ) in culture medium containing PI and images taken before ( A ; left) and after ( A ; right) 96 h. The call out box shows a representative ADMC being activated through loss of granularity over time. (Mag 20x). (B) Quantification of overall PI fluorescence before and after incubation. The percent of PI-positive cells was counted in culture. The * p = 0.0003 SK-BR-3 cell death at day 4 compared to day 0 when ADMC where sensitized with anti-HER2/ neu IgE. No cell death was observed with the NS IgE. (C) ADMC-induced breast cancer cell apoptosis. Anti-HER2/ neu IgE-sensitized ADMC (7.5 × 10 4 ) were incubated with SK-BR-3 (1 × 10 5 ) for 72 h, cytospins made, fixed, and incubated with Alexa Fluor TM 488 labeled, mouse anti-human tryptase (left; green) along with Alexa Fluor TM 647 labeled, mouse anti-human caspase 3 (red) or Alexa Fluor TM 647 labeled, isotype control IgG for caspase 3 Ab (right). (D) Quantification of overall Alexa Fluor TM 647 fluorescence before and after incubation. * p = 0.002 SK-BR-3 apoptosis comparing psIgE and anti-HER2/ neu IgE-sensitized cells at day 4 from three experiments. (E) ADMC killing of human breast cancer cells measured by caspase 3/7 activation Mitotracker™-red labeled ADMC (7.5 × 10 4 ; shown here as red cells) were sensitized with 1 μg/ml anti-HER2/ neu IgE (clone trastuzumab), washed, and incubated with caspase 3/7 green-labeled SK-BR-3 (10 5 and images taken before ( A , left) and after ( A ; right) 96 h. (F) Quantification of overall caspase 3/7 fluorescence before and after incubation. The percent of caspase 3/7-positive cells was counted in culture. * p = 0.004 SK-BR-3 cell death at day 4 compared to day 0 when ADMC where sensitized with anti-HER2/ neu IgE. No SK-BR-3 cell death was observed with the NS IgE.
    Figure Legend Snippet: ADMC killing of human breast cancer cells measured as evidence by the uptake of PI. (A) CellTracker™-red labeled ADMC (7.5 × 10 4 ; shown here as purple cells) were sensitized with 1 μg/ml anti-HER2/ neu IgE (clone trastuzumab), washed, and incubated with MitoTracker™-green-stained SK-BR-3 (10 5 ) in culture medium containing PI and images taken before ( A ; left) and after ( A ; right) 96 h. The call out box shows a representative ADMC being activated through loss of granularity over time. (Mag 20x). (B) Quantification of overall PI fluorescence before and after incubation. The percent of PI-positive cells was counted in culture. The * p = 0.0003 SK-BR-3 cell death at day 4 compared to day 0 when ADMC where sensitized with anti-HER2/ neu IgE. No cell death was observed with the NS IgE. (C) ADMC-induced breast cancer cell apoptosis. Anti-HER2/ neu IgE-sensitized ADMC (7.5 × 10 4 ) were incubated with SK-BR-3 (1 × 10 5 ) for 72 h, cytospins made, fixed, and incubated with Alexa Fluor TM 488 labeled, mouse anti-human tryptase (left; green) along with Alexa Fluor TM 647 labeled, mouse anti-human caspase 3 (red) or Alexa Fluor TM 647 labeled, isotype control IgG for caspase 3 Ab (right). (D) Quantification of overall Alexa Fluor TM 647 fluorescence before and after incubation. * p = 0.002 SK-BR-3 apoptosis comparing psIgE and anti-HER2/ neu IgE-sensitized cells at day 4 from three experiments. (E) ADMC killing of human breast cancer cells measured by caspase 3/7 activation Mitotracker™-red labeled ADMC (7.5 × 10 4 ; shown here as red cells) were sensitized with 1 μg/ml anti-HER2/ neu IgE (clone trastuzumab), washed, and incubated with caspase 3/7 green-labeled SK-BR-3 (10 5 and images taken before ( A , left) and after ( A ; right) 96 h. (F) Quantification of overall caspase 3/7 fluorescence before and after incubation. The percent of caspase 3/7-positive cells was counted in culture. * p = 0.004 SK-BR-3 cell death at day 4 compared to day 0 when ADMC where sensitized with anti-HER2/ neu IgE. No SK-BR-3 cell death was observed with the NS IgE.

    Techniques Used: Labeling, Incubation, Staining, Fluorescence, Activation Assay

    ADMC functional response. Human skin MC (black box) or ADMC (gray box; 10 6 ) were challenged with or without (spontaneous release) 1 μg/ml anti-FcεRI Abs or anti-NP IgE + antigen (IgE-Ag) and degranulation (A) or GM-CSF production (B) assessed in the supernatants. Error bars represent ± SD. * p
    Figure Legend Snippet: ADMC functional response. Human skin MC (black box) or ADMC (gray box; 10 6 ) were challenged with or without (spontaneous release) 1 μg/ml anti-FcεRI Abs or anti-NP IgE + antigen (IgE-Ag) and degranulation (A) or GM-CSF production (B) assessed in the supernatants. Error bars represent ± SD. * p

    Techniques Used: Functional Assay

    Time lapse, confocal microscopy of ADMC binding to breast cancer cells. ADMC (10 5 -10 6 ) were sensitized with 1 μg/ml of trastuzumab IgE ( A , 20X) or NS IgE ( B , 40X) followed by MitoTracker™ Green. The MitoTracker™ Green-loaded ADMC were added to adherent SK-BR-3 (10 5 -10 6 ) that had been pre-stained with MitoTracker™ Red and time lapse video taken over 6 h. The white circular boundaries and arrows represent starting point and tracking of ADMC (green) at time 0 to SK-BR-3 (red) binding over the 6 h. 20X magnification was used to capture the cellular tracking (start and stop) that can be observed in accompanying video.
    Figure Legend Snippet: Time lapse, confocal microscopy of ADMC binding to breast cancer cells. ADMC (10 5 -10 6 ) were sensitized with 1 μg/ml of trastuzumab IgE ( A , 20X) or NS IgE ( B , 40X) followed by MitoTracker™ Green. The MitoTracker™ Green-loaded ADMC were added to adherent SK-BR-3 (10 5 -10 6 ) that had been pre-stained with MitoTracker™ Red and time lapse video taken over 6 h. The white circular boundaries and arrows represent starting point and tracking of ADMC (green) at time 0 to SK-BR-3 (red) binding over the 6 h. 20X magnification was used to capture the cellular tracking (start and stop) that can be observed in accompanying video.

    Techniques Used: Confocal Microscopy, Binding Assay, Staining

    Breast cancer cell-induced ADMC mediator release. ADMC were sensitized with 1 μg/ml anti-HER/ neu IgE (clone C6MH3-B1 IgE or trastuzumab IgE), washed, and incubated with SK-BR-3 cells and degranulation (A) or cytokine release (B) assessed. Data are from a single experiment representative of experiments performed on cells derived from four separate donors. Error bars represent ± SD. * p
    Figure Legend Snippet: Breast cancer cell-induced ADMC mediator release. ADMC were sensitized with 1 μg/ml anti-HER/ neu IgE (clone C6MH3-B1 IgE or trastuzumab IgE), washed, and incubated with SK-BR-3 cells and degranulation (A) or cytokine release (B) assessed. Data are from a single experiment representative of experiments performed on cells derived from four separate donors. Error bars represent ± SD. * p

    Techniques Used: Incubation, Derivative Assay

    Mediators from FcεRI-challenged ADMC induce SK-BR-3 cell killing. (A) ADMC (1.3 × 10 6 ) were challenged with optimal concentrations of anti-FcεRI stimuli (70% release) for 24 h and supernatants (XL media; no cells) from these ADMC were incubated with the MitoTracker™ green-stained SK-BR-3 (10 5 ) in culture medium containing optimal concentrations of PI and images taken before (left) and after (right) 96 h. (B) Quantification of overall PI fluorescence before and after incubation. The increased number of red cells indicates breast cancer cell death as indicated by the PI (red) and quantified in showing overall PI fluorescence before and after incubation. Graph represents average PI intensity from two separate experiments (±SD; * p = 0.0008). (C) Mediators from FcεRI-challenged ADMC induce human breast cancer cell apoptosis. The same media from anti-FcεRI challenged ADMC were incubated with SK-BR-3 (10 5 ) for 72 h, cytospins prepared, fixed, and incubated with Alexa Fluor 647 labeled, anti-human caspase 3 (left) or Alexa Fluor 647 labeled, isotype control Ab for caspase 3 (right). Representative panels are shown. (D) Quantification of overall Alexa Fluor TM 647 fluorescence before and after incubation with supernatants from FcεRI activated ADMC. * p = 0.01 SK-BR-3 apoptosis comparing anti-HER2/ neu IgE-sensitized cells at day 0 vs. day 4 from three experiments. (E) Mediators from FcεRI-challenged ADMC induce SK-BR-3 cell killing measured by caspase 3/7. ADMC (1.8 × 10 6 ) were challenged with optimal concentrations of anti-FcεRI stimuli (63% release) for 24 h and supernatants (no cells) from these ADMC were incubated with SK-BR-3 (10 5 ) and caspase 3/7 green images taken before (left) and after (right) 96 h. The increased number of green cells indicates breast cancer cell death as indicated by the caspase 3/7 and quantified by counting live vs. dead cells before and after incubation. (F) Graph represents average percentage of cells from two separate experiments (±SD; * p = 0.002). (G) Blocking TNF-α significantly reduces SK-BR-3 cell death. SK-BR-3 were treated and quantified as in (C) except anti-TNF-α Ab were added during the incubation time. * p = 0.028 Decrease in SK-BR-3 cell death when anti-TNF-α Ab are added to the supernatants from anti-FcεRI stimulated ADMC.
    Figure Legend Snippet: Mediators from FcεRI-challenged ADMC induce SK-BR-3 cell killing. (A) ADMC (1.3 × 10 6 ) were challenged with optimal concentrations of anti-FcεRI stimuli (70% release) for 24 h and supernatants (XL media; no cells) from these ADMC were incubated with the MitoTracker™ green-stained SK-BR-3 (10 5 ) in culture medium containing optimal concentrations of PI and images taken before (left) and after (right) 96 h. (B) Quantification of overall PI fluorescence before and after incubation. The increased number of red cells indicates breast cancer cell death as indicated by the PI (red) and quantified in showing overall PI fluorescence before and after incubation. Graph represents average PI intensity from two separate experiments (±SD; * p = 0.0008). (C) Mediators from FcεRI-challenged ADMC induce human breast cancer cell apoptosis. The same media from anti-FcεRI challenged ADMC were incubated with SK-BR-3 (10 5 ) for 72 h, cytospins prepared, fixed, and incubated with Alexa Fluor 647 labeled, anti-human caspase 3 (left) or Alexa Fluor 647 labeled, isotype control Ab for caspase 3 (right). Representative panels are shown. (D) Quantification of overall Alexa Fluor TM 647 fluorescence before and after incubation with supernatants from FcεRI activated ADMC. * p = 0.01 SK-BR-3 apoptosis comparing anti-HER2/ neu IgE-sensitized cells at day 0 vs. day 4 from three experiments. (E) Mediators from FcεRI-challenged ADMC induce SK-BR-3 cell killing measured by caspase 3/7. ADMC (1.8 × 10 6 ) were challenged with optimal concentrations of anti-FcεRI stimuli (63% release) for 24 h and supernatants (no cells) from these ADMC were incubated with SK-BR-3 (10 5 ) and caspase 3/7 green images taken before (left) and after (right) 96 h. The increased number of green cells indicates breast cancer cell death as indicated by the caspase 3/7 and quantified by counting live vs. dead cells before and after incubation. (F) Graph represents average percentage of cells from two separate experiments (±SD; * p = 0.002). (G) Blocking TNF-α significantly reduces SK-BR-3 cell death. SK-BR-3 were treated and quantified as in (C) except anti-TNF-α Ab were added during the incubation time. * p = 0.028 Decrease in SK-BR-3 cell death when anti-TNF-α Ab are added to the supernatants from anti-FcεRI stimulated ADMC.

    Techniques Used: Incubation, Staining, Fluorescence, Labeling, Blocking Assay

    5) Product Images from "Rapid and accurate analysis of stem cell-derived extracellular vesicles with super resolution microscopy and live imaging"

    Article Title: Rapid and accurate analysis of stem cell-derived extracellular vesicles with super resolution microscopy and live imaging

    Journal: Biochimica et Biophysica Acta. Molecular Cell Research

    doi: 10.1016/j.bbamcr.2018.09.008

    d-STORM imaging of DiD-labelled EVs isolated from MSCs. (a) Graphical representation of the sample scanning pattern with snapshots of areas 1, 2 and 3 recorded in TIRF. Scale bar: 2 μm. (b–f) d-STORM images of MSC-derived EVs showing (b) TIRF (white), (c) rSWF (sum of wide field frames over the experiment) (green 75), (d) d-STORM (magenta) and (e) merge of rSWF and d-STORM. Scale bar: 500 nm. (f) Cross-sectional line-profiling of 2 EVs imaged in TIRF, rSWF and d-STORM as shown in (b) to (e), measured across the line indicated as dotted arrow in (b). (g) Absolute frequency of photon number recorded in [DiD in PBS] (green), [DiD in Exo-E medium] (red), [DiD in serum-free Exo-E medium] (purple) and DiD-labelled EV sample (blue).
    Figure Legend Snippet: d-STORM imaging of DiD-labelled EVs isolated from MSCs. (a) Graphical representation of the sample scanning pattern with snapshots of areas 1, 2 and 3 recorded in TIRF. Scale bar: 2 μm. (b–f) d-STORM images of MSC-derived EVs showing (b) TIRF (white), (c) rSWF (sum of wide field frames over the experiment) (green 75), (d) d-STORM (magenta) and (e) merge of rSWF and d-STORM. Scale bar: 500 nm. (f) Cross-sectional line-profiling of 2 EVs imaged in TIRF, rSWF and d-STORM as shown in (b) to (e), measured across the line indicated as dotted arrow in (b). (g) Absolute frequency of photon number recorded in [DiD in PBS] (green), [DiD in Exo-E medium] (red), [DiD in serum-free Exo-E medium] (purple) and DiD-labelled EV sample (blue).

    Techniques Used: Imaging, Isolation, Derivative Assay

    6) Product Images from "C-BERST: Defining subnuclear proteomic landscapes at genomic elements with dCas9-APEX2"

    Article Title: C-BERST: Defining subnuclear proteomic landscapes at genomic elements with dCas9-APEX2

    Journal: Nature methods

    doi: 10.1038/s41592-018-0006-2

    Successful capture of alpha-satellite-associated proteomes in live human cells by C-BERST. ( a ) Ratiometric C-BERST (using SILAC) was used to profile the alpha-satellite-associated proteome. A volcano plot of C-BERST-labeled, centromere-associated proteins in U2OS cells is shown. For each protein, the H/M SILAC ratio reflects the enrichment of identified proteins in sgAlpha vs. sgNS cells. 1,134 proteins (indicated by blue and red dots) are statistically enriched [BH-adjusted p value
    Figure Legend Snippet: Successful capture of alpha-satellite-associated proteomes in live human cells by C-BERST. ( a ) Ratiometric C-BERST (using SILAC) was used to profile the alpha-satellite-associated proteome. A volcano plot of C-BERST-labeled, centromere-associated proteins in U2OS cells is shown. For each protein, the H/M SILAC ratio reflects the enrichment of identified proteins in sgAlpha vs. sgNS cells. 1,134 proteins (indicated by blue and red dots) are statistically enriched [BH-adjusted p value

    Techniques Used: Labeling

    Using C-BERST to biotinylate telomere-associated proteins in living human cells. ( a ) Diagram of the C-BERST workflow. ( b ) Telomere-associated proteome identification by ratiometric C-BERST. A volcano plot is shown for C-BERST-labeled, telomere-associated proteins in U2OS cells. For each protein, the H/M SILAC ratio reflects the enrichment of identified proteins in sgTelo vs. sgNS cells. 359 proteins (indicated by blue and red dots) are statistically enriched [BH-adjusted p value
    Figure Legend Snippet: Using C-BERST to biotinylate telomere-associated proteins in living human cells. ( a ) Diagram of the C-BERST workflow. ( b ) Telomere-associated proteome identification by ratiometric C-BERST. A volcano plot is shown for C-BERST-labeled, telomere-associated proteins in U2OS cells. For each protein, the H/M SILAC ratio reflects the enrichment of identified proteins in sgTelo vs. sgNS cells. 359 proteins (indicated by blue and red dots) are statistically enriched [BH-adjusted p value

    Techniques Used: Labeling

    7) Product Images from "The mature activating natural killer cell immunologic synapse is formed in distinct stages"

    Article Title: The mature activating natural killer cell immunologic synapse is formed in distinct stages

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1835830100

    CD2 and F-actin accumulate at the activating NKIS more readily than does perforin. ( A ) Conjugation of YTS-CD2/GFP cells with 721.221 over time (⋄) was calculated as a percentage of total YTS-CD2/GFP cells present. ( B ) The accumulation of F-actin (▴), CD2/GFP (▪), and perforin (•) at the IS of YTS-CD2/GFP cells conjugated with 721.221 cells was evaluated for each time point (except time 0 due to the insignificant number of conjugates found). Individual points are the mean of four to eight experiments ± SD.
    Figure Legend Snippet: CD2 and F-actin accumulate at the activating NKIS more readily than does perforin. ( A ) Conjugation of YTS-CD2/GFP cells with 721.221 over time (⋄) was calculated as a percentage of total YTS-CD2/GFP cells present. ( B ) The accumulation of F-actin (▴), CD2/GFP (▪), and perforin (•) at the IS of YTS-CD2/GFP cells conjugated with 721.221 cells was evaluated for each time point (except time 0 due to the insignificant number of conjugates found). Individual points are the mean of four to eight experiments ± SD.

    Techniques Used: Conjugation Assay

    Cumulative summary of molecular polarization to the activating NKIS. Accumulation of F-actin (white bars), CD2, CD2/GFP, CD11a, or CD11b and perforin (gray bars) at the activating NKIS in ex vivo NK cells conjugated with K562 cells, or in YTS-CD2/GFP cells conjugated with 721.221 cells are shown. Cells were untreated, DMSO-treated, Cyt-D-treated, from a WAS patient, or colchicine-treated, as displayed on the x axis. Each cluster of bars represents the mean ± SD of three to six independent experiments, and the entire figure summarizes ≈4,000 individual conjugates. In all untreated samples, perforin was polarized less frequently than F-actin or the respective cell-surface receptor ( P
    Figure Legend Snippet: Cumulative summary of molecular polarization to the activating NKIS. Accumulation of F-actin (white bars), CD2, CD2/GFP, CD11a, or CD11b and perforin (gray bars) at the activating NKIS in ex vivo NK cells conjugated with K562 cells, or in YTS-CD2/GFP cells conjugated with 721.221 cells are shown. Cells were untreated, DMSO-treated, Cyt-D-treated, from a WAS patient, or colchicine-treated, as displayed on the x axis. Each cluster of bars represents the mean ± SD of three to six independent experiments, and the entire figure summarizes ≈4,000 individual conjugates. In all untreated samples, perforin was polarized less frequently than F-actin or the respective cell-surface receptor ( P

    Techniques Used: Ex Vivo, Cell Surface Receptor Assay

    F-actin with CD2, CD11a, or CD11b accumulate in the pSMAC and perforin in the cSMAC of the mature lytic NKIS. Representative lytic synapses were evaluated throughout their volume, and the z , x plane was reconstructed. The first three columns show lytic synapses in ex vivo NK cells conjugated with K562 cells. F-actin (blue, top row), perforin (green, second row), or CD2 in the first column, CD11a in the second column, or CD11b in the third column (red, third row) are shown. A synapse between a YTS-CD2/GFP cell and 721.221 cell is in the fourth column with F-actin (blue), perforin (red), and CD2/GFP (green) being shown. A merged overlay of all fluorescent channels is shown in the bottom row. (Scale bars, 5 μm). The mean frequency of synapses with an F-actin and CD2, CD11a, or CD11b in the pSMAC and perforin in the cSMAC are above the representative images ( n = 3–5 experiments/donors ± SD).
    Figure Legend Snippet: F-actin with CD2, CD11a, or CD11b accumulate in the pSMAC and perforin in the cSMAC of the mature lytic NKIS. Representative lytic synapses were evaluated throughout their volume, and the z , x plane was reconstructed. The first three columns show lytic synapses in ex vivo NK cells conjugated with K562 cells. F-actin (blue, top row), perforin (green, second row), or CD2 in the first column, CD11a in the second column, or CD11b in the third column (red, third row) are shown. A synapse between a YTS-CD2/GFP cell and 721.221 cell is in the fourth column with F-actin (blue), perforin (red), and CD2/GFP (green) being shown. A merged overlay of all fluorescent channels is shown in the bottom row. (Scale bars, 5 μm). The mean frequency of synapses with an F-actin and CD2, CD11a, or CD11b in the pSMAC and perforin in the cSMAC are above the representative images ( n = 3–5 experiments/donors ± SD).

    Techniques Used: Ex Vivo

    F-actin, along with CD2, CD11a, CD11b, and perforin, all polarize to the mature lytic NKIS. Ex vivo NK cells (small cells) are shown conjugated with K562 cells (large cells) in the top three rows, and a YTS-CD2/GFP cell (top cell) with a 721.221 cell (bottom cell) in the bottom row. Differential interference contrast images are in the left column and fluorescent images are in the right columns. F-actin is in the second column (blue). CD2, CD11a, or CD11b (red) and CD2/GFP (green) are in the third column. Perforin (green) in ex vivo NK cells and in YTS-CD2/GFP cells (red) is in the fourth column. A merged overlay of all fluorescent channels is in the fifth column.
    Figure Legend Snippet: F-actin, along with CD2, CD11a, CD11b, and perforin, all polarize to the mature lytic NKIS. Ex vivo NK cells (small cells) are shown conjugated with K562 cells (large cells) in the top three rows, and a YTS-CD2/GFP cell (top cell) with a 721.221 cell (bottom cell) in the bottom row. Differential interference contrast images are in the left column and fluorescent images are in the right columns. F-actin is in the second column (blue). CD2, CD11a, or CD11b (red) and CD2/GFP (green) are in the third column. Perforin (green) in ex vivo NK cells and in YTS-CD2/GFP cells (red) is in the fourth column. A merged overlay of all fluorescent channels is in the fifth column.

    Techniques Used: Ex Vivo

    8) Product Images from "Inverse Agonist and Pharmacochaperone Properties of MK-0524 on the Prostanoid DP1 Receptor"

    Article Title: Inverse Agonist and Pharmacochaperone Properties of MK-0524 on the Prostanoid DP1 Receptor

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0065767

    MK-0524 promotes cell surface expression of DP1. Cell surface expression of the receptor was measured by ELISA as described under “ Materials and Methods ” in HEK293 cells transiently expressing Flag-DP1 incubated for the indicated times with 1 µM of PGD 2 , BW245C, BWA868C or MK-0524. The results are shown as the percentage of cell surface expression of Flag-DP1 in cells stimulated with the ligands compared with cells treated with control vehicles. Results are the mean ± S.E. of at least four independent experiments.
    Figure Legend Snippet: MK-0524 promotes cell surface expression of DP1. Cell surface expression of the receptor was measured by ELISA as described under “ Materials and Methods ” in HEK293 cells transiently expressing Flag-DP1 incubated for the indicated times with 1 µM of PGD 2 , BW245C, BWA868C or MK-0524. The results are shown as the percentage of cell surface expression of Flag-DP1 in cells stimulated with the ligands compared with cells treated with control vehicles. Results are the mean ± S.E. of at least four independent experiments.

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Incubation

    MK-0524 does not modulate ERK1/2 activation by DP1. HEK293 cells transiently expressing Flag-DP1 were stimulated with 1 µM PGD 2 or MK-0524 for the indicated times. ERK1/2 activation was analyzed by Western blot using a phospho-ERK1/2 (pERK1/2) antibody as described under “ Materials and Methods ”. Total amounts of ERK1/2 in the loaded samples were revealed by an anti-ERK1/2 antibody. The blots shown are representative of three separate experiments. Densitometry analyses (pERK1/2/ERK) of at least three different experiments were performed. IB , immunoblotting. * is P
    Figure Legend Snippet: MK-0524 does not modulate ERK1/2 activation by DP1. HEK293 cells transiently expressing Flag-DP1 were stimulated with 1 µM PGD 2 or MK-0524 for the indicated times. ERK1/2 activation was analyzed by Western blot using a phospho-ERK1/2 (pERK1/2) antibody as described under “ Materials and Methods ”. Total amounts of ERK1/2 in the loaded samples were revealed by an anti-ERK1/2 antibody. The blots shown are representative of three separate experiments. Densitometry analyses (pERK1/2/ERK) of at least three different experiments were performed. IB , immunoblotting. * is P

    Techniques Used: Activation Assay, Expressing, Western Blot

    MK-0524 reduces DP1 cAMP signaling below basal levels. HEK293 cells transiently expressing Flag-DP1 were stimulated with 10 nM (A) or increasing concentrations (B) of the indicated ligands for 10 min and cAMP levels were measured as described in “ Materials and Methods ”. Results are presented in fmol/well above or below basal cAMP production by DP1 in absence of ligand (set at 0) (A) or as the % of the maximal response obtained with PGD 2 stimulation (B). Data are the mean ± S.E. of at least three independent experiments. *** is P
    Figure Legend Snippet: MK-0524 reduces DP1 cAMP signaling below basal levels. HEK293 cells transiently expressing Flag-DP1 were stimulated with 10 nM (A) or increasing concentrations (B) of the indicated ligands for 10 min and cAMP levels were measured as described in “ Materials and Methods ”. Results are presented in fmol/well above or below basal cAMP production by DP1 in absence of ligand (set at 0) (A) or as the % of the maximal response obtained with PGD 2 stimulation (B). Data are the mean ± S.E. of at least three independent experiments. *** is P

    Techniques Used: Expressing

    The MK-0524-mediated reduction in DP1 cAMP signaling below basal levels is unaffected by pertussis toxin. HEK293 cells transiently expressing Flag-DP1 pretreated or not with 1 µg/ml of pertussis toxin (PTX) for 10 min were incubated with increasing concentrations of MK-0524 and cAMP levels were measured as described in “ Materials and Methods ”. Results are presented as fmol of cAMP generated per well. Data are the mean ± S.E. of at least three independent experiments.
    Figure Legend Snippet: The MK-0524-mediated reduction in DP1 cAMP signaling below basal levels is unaffected by pertussis toxin. HEK293 cells transiently expressing Flag-DP1 pretreated or not with 1 µg/ml of pertussis toxin (PTX) for 10 min were incubated with increasing concentrations of MK-0524 and cAMP levels were measured as described in “ Materials and Methods ”. Results are presented as fmol of cAMP generated per well. Data are the mean ± S.E. of at least three independent experiments.

    Techniques Used: Expressing, Incubation, Generated

    The total expression of the DP1 protein is not modulated by MK-0524. Lysates of HEK293 cells transiently expressing Flag-DP1 incubated for 24 h with vehicle or 1 µM of MK-0524 were analyzed by Western blot using a monoclonal Flag antibody. The blot shown is representative of three separate experiments. IB , immunoblotting.
    Figure Legend Snippet: The total expression of the DP1 protein is not modulated by MK-0524. Lysates of HEK293 cells transiently expressing Flag-DP1 incubated for 24 h with vehicle or 1 µM of MK-0524 were analyzed by Western blot using a monoclonal Flag antibody. The blot shown is representative of three separate experiments. IB , immunoblotting.

    Techniques Used: Expressing, Incubation, Western Blot

    The promotion of DP1 cell surface targeting by MK-0524 is inhibited by Brefeldin A. Cell surface receptor expression was measured by ELISA as described under “ Materials and Methods ” in HEK293 cells transiently expressing Flag-DP1 that were pre-incubated with vehicle or 20 µM of Brefeldin A (BFA) for 30 min, and then treated with 1 µM of MK-0524 or its control vehicle for 90 min. The results are shown as the percentage increase of DP1 cell surface expression when compared to control cells treated with vehicle. Results are the mean ± S.E. of three independent experiments. * is P
    Figure Legend Snippet: The promotion of DP1 cell surface targeting by MK-0524 is inhibited by Brefeldin A. Cell surface receptor expression was measured by ELISA as described under “ Materials and Methods ” in HEK293 cells transiently expressing Flag-DP1 that were pre-incubated with vehicle or 20 µM of Brefeldin A (BFA) for 30 min, and then treated with 1 µM of MK-0524 or its control vehicle for 90 min. The results are shown as the percentage increase of DP1 cell surface expression when compared to control cells treated with vehicle. Results are the mean ± S.E. of three independent experiments. * is P

    Techniques Used: Cell Surface Receptor Assay, Expressing, Enzyme-linked Immunosorbent Assay, Incubation

    DP1 does not undergo constitutive internalization. Cell surface receptor expression was measured by ELISA as described under “ Materials and Methods ” in HEK293 transiently co-transfected with pcDNA3-Flag-DP1 or pcDNA3-Flag-TPβ in combination with pcDNA3 or pcDNA3-dynamin-K44A, a dominant negative mutant of dynamin. Data are shown as the percentage of cell surface receptor expression in cells co-transfected with pcDNA3 for each receptor. Results are the mean ± S.E. of at least three independent experiments. ** is P
    Figure Legend Snippet: DP1 does not undergo constitutive internalization. Cell surface receptor expression was measured by ELISA as described under “ Materials and Methods ” in HEK293 transiently co-transfected with pcDNA3-Flag-DP1 or pcDNA3-Flag-TPβ in combination with pcDNA3 or pcDNA3-dynamin-K44A, a dominant negative mutant of dynamin. Data are shown as the percentage of cell surface receptor expression in cells co-transfected with pcDNA3 for each receptor. Results are the mean ± S.E. of at least three independent experiments. ** is P

    Techniques Used: Cell Surface Receptor Assay, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Dominant Negative Mutation

    MK-0524 induces translocation of DP1 from intracellular compartments to the plasma membrane. The distribution of DP1 in HEK293 cells was determined by immunofluorescence confocal microscopy. HEK293 cells transfected with Flag-DP1 were treated with vehicle or 1 µM of MK-0524 alone or in presence of 20 µM Brefeldin A for 90 min. Cells were labeled with mouse anti-FLAG and either a rabbit anti-calnexin antibody (top and midlle panels) or a rabbit anti-protein disulfide isomerase (PDI) antibody (lower panel) as described under “ Materials and Methods ”. Secondary antibodies were Alexa Fluor 488 donkey anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG. Merge images of the green-labelled DP1 and red-labeled calnexin or PDI are shown. Bars, 10 µM.
    Figure Legend Snippet: MK-0524 induces translocation of DP1 from intracellular compartments to the plasma membrane. The distribution of DP1 in HEK293 cells was determined by immunofluorescence confocal microscopy. HEK293 cells transfected with Flag-DP1 were treated with vehicle or 1 µM of MK-0524 alone or in presence of 20 µM Brefeldin A for 90 min. Cells were labeled with mouse anti-FLAG and either a rabbit anti-calnexin antibody (top and midlle panels) or a rabbit anti-protein disulfide isomerase (PDI) antibody (lower panel) as described under “ Materials and Methods ”. Secondary antibodies were Alexa Fluor 488 donkey anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG. Merge images of the green-labelled DP1 and red-labeled calnexin or PDI are shown. Bars, 10 µM.

    Techniques Used: Translocation Assay, Immunofluorescence, Confocal Microscopy, Transfection, Labeling

    MK-0524 promotes the interaction between DP1 and the ANKRD13C protein. HEK293 cells transiently co-expressing Flag-DP1 and ANKRD13C-myc were treated with vehicle (ethanol) or 1 µM MK-0524 for 24 h. Flag-DP1 was immunoprecipitated as described under “ Materials and Methods ” and immunoprecipitated samples as well as cell lysates were analyzed by Western blot with anti-Flag and anti-Myc antibodies. The blots shown are representative of three separate experiments. The ratio of the amount of ANKRD13C that was co-immunoprecipitated on the quantity of receptor immunoprecipitated was calculated by densitometry analyses from the three separate experiments and the results are shown in the bottom panel as the mean ± S.E. * is P
    Figure Legend Snippet: MK-0524 promotes the interaction between DP1 and the ANKRD13C protein. HEK293 cells transiently co-expressing Flag-DP1 and ANKRD13C-myc were treated with vehicle (ethanol) or 1 µM MK-0524 for 24 h. Flag-DP1 was immunoprecipitated as described under “ Materials and Methods ” and immunoprecipitated samples as well as cell lysates were analyzed by Western blot with anti-Flag and anti-Myc antibodies. The blots shown are representative of three separate experiments. The ratio of the amount of ANKRD13C that was co-immunoprecipitated on the quantity of receptor immunoprecipitated was calculated by densitometry analyses from the three separate experiments and the results are shown in the bottom panel as the mean ± S.E. * is P

    Techniques Used: Expressing, Immunoprecipitation, Western Blot

    9) Product Images from "Release of a Wound-Healing Agent from PLGA Microspheres in a Thermosensitive Gel"

    Article Title: Release of a Wound-Healing Agent from PLGA Microspheres in a Thermosensitive Gel

    Journal: BioMed Research International

    doi: 10.1155/2013/387863

    KSL-W standard curves (280 nm). (a) high-performance liquid chromatography; (b) UV spectrophotometry. Readings were obtained from samples determined in triplicates (mean ± standard errors).
    Figure Legend Snippet: KSL-W standard curves (280 nm). (a) high-performance liquid chromatography; (b) UV spectrophotometry. Readings were obtained from samples determined in triplicates (mean ± standard errors).

    Techniques Used: High Performance Liquid Chromatography, Spectrophotometry

    Bactericidal activity of fractions collected from KSL-W PLGA microsphere formulation release using the broth microdilution method. The MIC of KSL-W peptide alone against S. epidermidis was 6.25 μ g/mL.
    Figure Legend Snippet: Bactericidal activity of fractions collected from KSL-W PLGA microsphere formulation release using the broth microdilution method. The MIC of KSL-W peptide alone against S. epidermidis was 6.25 μ g/mL.

    Techniques Used: Activity Assay

    KSL-W PLGA microspheres particle size and particle size distribution using Smart Tiff from Zeiss; n = 30. Average means; Formulation A, 46.4 μ m ± 17.9 μ m; Formulation B, 25.8 μ m ± 14.1 μ m; Formulation C, 40.0 μ m ± 12.8 μ m.
    Figure Legend Snippet: KSL-W PLGA microspheres particle size and particle size distribution using Smart Tiff from Zeiss; n = 30. Average means; Formulation A, 46.4 μ m ± 17.9 μ m; Formulation B, 25.8 μ m ± 14.1 μ m; Formulation C, 40.0 μ m ± 12.8 μ m.

    Techniques Used:

    Schematic of the double-emulsification solvent extraction/evaporation method for the preparation of KSL-W-PLGA microspheres. Polycarbonate (PC) membrane filter (1 μ m size cutoff) was used to filter the MS.
    Figure Legend Snippet: Schematic of the double-emulsification solvent extraction/evaporation method for the preparation of KSL-W-PLGA microspheres. Polycarbonate (PC) membrane filter (1 μ m size cutoff) was used to filter the MS.

    Techniques Used: Evaporation, Mass Spectrometry

    High-performance liquid chromatogram of KSL-W (0.5 mg/mL) in DI water.
    Figure Legend Snippet: High-performance liquid chromatogram of KSL-W (0.5 mg/mL) in DI water.

    Techniques Used:

    10) Product Images from "Siramesine triggers cell death through destabilisation of mitochondria, but not lysosomes"

    Article Title: Siramesine triggers cell death through destabilisation of mitochondria, but not lysosomes

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.361

    Siramesine-induced changes of cathepsin L and LC3 at a protein level. ( a ) HaCaT cell were treated with different siramesine concentrations, and the total cell extracts were prepared in RIPA buffer at the indicated time points. Proteins were resolved in 12.5% SDS-PAGE and transferred to a nitrocellulose membrane. Cathepsin L and LC3 were labelled with specific antibodies. ( b ) Immunocytochemistry of HaCaT cells treated with different concentrations of siramesine labelled with LC3 (green) and LAMP-2 A antibodies (red). S, siramesine
    Figure Legend Snippet: Siramesine-induced changes of cathepsin L and LC3 at a protein level. ( a ) HaCaT cell were treated with different siramesine concentrations, and the total cell extracts were prepared in RIPA buffer at the indicated time points. Proteins were resolved in 12.5% SDS-PAGE and transferred to a nitrocellulose membrane. Cathepsin L and LC3 were labelled with specific antibodies. ( b ) Immunocytochemistry of HaCaT cells treated with different concentrations of siramesine labelled with LC3 (green) and LAMP-2 A antibodies (red). S, siramesine

    Techniques Used: SDS Page, Immunocytochemistry

    11) Product Images from "Protein Phosphatases Decrease Their Activity during Capacitation: A New Requirement for This Event"

    Article Title: Protein Phosphatases Decrease Their Activity during Capacitation: A New Requirement for This Event

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0081286

    Effect of phosphatase inhibitors on PP1, PP2A, and PP2B enzymatic activity. Sperm were selected and re-suspended in NCM. Each phosphatase activity was measured in sperm extracts as indicated in Materials and Methods in the absence of inhibitors (black bars) and then in the presence of 10 nM okadaic acid (white bar); 40 µg/ml I2 (grey bar); 90 nM endothall (dotted bar); 0.1 nM okadaic acid (light grey bar); or 0.1 nM deltamethrin (striped bar). Results were obtained from five different donors and are expressed as the mean±SEM of forty measurements every 2 min. * p
    Figure Legend Snippet: Effect of phosphatase inhibitors on PP1, PP2A, and PP2B enzymatic activity. Sperm were selected and re-suspended in NCM. Each phosphatase activity was measured in sperm extracts as indicated in Materials and Methods in the absence of inhibitors (black bars) and then in the presence of 10 nM okadaic acid (white bar); 40 µg/ml I2 (grey bar); 90 nM endothall (dotted bar); 0.1 nM okadaic acid (light grey bar); or 0.1 nM deltamethrin (striped bar). Results were obtained from five different donors and are expressed as the mean±SEM of forty measurements every 2 min. * p

    Techniques Used: Activity Assay

    Effect of protein phosphatase inhibitors on human sperm capacitation. Human sperm were incubated in NCM (A) or RCM (B) for 120 min, and the capacitation pattern was evaluated using the CTC assay, as described in the Materials and Methods . The inhibitors used were okadaic acid (black squares), deltamethrin (green diamonds), and endothall (blue triangles). Results were obtained from five different donors and were expressed as the mean±SEM of the percentage of B pattern. The arrow indicates the addition of BSA and bicarbonate. * p
    Figure Legend Snippet: Effect of protein phosphatase inhibitors on human sperm capacitation. Human sperm were incubated in NCM (A) or RCM (B) for 120 min, and the capacitation pattern was evaluated using the CTC assay, as described in the Materials and Methods . The inhibitors used were okadaic acid (black squares), deltamethrin (green diamonds), and endothall (blue triangles). Results were obtained from five different donors and were expressed as the mean±SEM of the percentage of B pattern. The arrow indicates the addition of BSA and bicarbonate. * p

    Techniques Used: Incubation

    Effect of phosphatase inhibitors on phosphorylation of threonine residues of human sperm proteins. Sperm were selected in NCM and then re-suspended in RCM in the absence (RCM) or in the presence of 10 nM okadaic acid (OA), 90 nM endothall (E), or 0.1 nM deltamethrin (D), for 1 min. Then, the samples were processed for western blot analysis with a monoclonal anti-p-Thr antibody. Molecular mass of selected bands is indicated at the right. The lower panels indicate load control with anti β-tubulin antibody. The immunoblots shown are representative of three different experiments with three different donors.
    Figure Legend Snippet: Effect of phosphatase inhibitors on phosphorylation of threonine residues of human sperm proteins. Sperm were selected in NCM and then re-suspended in RCM in the absence (RCM) or in the presence of 10 nM okadaic acid (OA), 90 nM endothall (E), or 0.1 nM deltamethrin (D), for 1 min. Then, the samples were processed for western blot analysis with a monoclonal anti-p-Thr antibody. Molecular mass of selected bands is indicated at the right. The lower panels indicate load control with anti β-tubulin antibody. The immunoblots shown are representative of three different experiments with three different donors.

    Techniques Used: Western Blot

    12) Product Images from "Use of a Molecular Decoy to Segregate Transport from Antigenicity in the FrpB Iron Transporter from Neisseria meningitidis"

    Article Title: Use of a Molecular Decoy to Segregate Transport from Antigenicity in the FrpB Iron Transporter from Neisseria meningitidis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056746

    Structural changes on binding of Fe to FrpB F5-1. (A) External loop movement during Fe recognition. The structure of FrpB F5-1 with Fe bound is shown in red, superimposed on the apoprotein form in green. The locations of Arg293, Arg298 and His573 are shown relative to the bound Fe atom. (B) Change in the conformation of the N-terminus as a result of Fe binding. Part of the β-barrel has been removed, for clarity. Colors used are as for part (A).
    Figure Legend Snippet: Structural changes on binding of Fe to FrpB F5-1. (A) External loop movement during Fe recognition. The structure of FrpB F5-1 with Fe bound is shown in red, superimposed on the apoprotein form in green. The locations of Arg293, Arg298 and His573 are shown relative to the bound Fe atom. (B) Change in the conformation of the N-terminus as a result of Fe binding. Part of the β-barrel has been removed, for clarity. Colors used are as for part (A).

    Techniques Used: Binding Assay

    Structure and alignment of the FrpB hypervariable sequence regions. (A) Superposition of two hypervariable regions F5-1 purple, F3-3 (A chain) green. (B) Sequence alignment of the HR regions from the major FrpB variants.
    Figure Legend Snippet: Structure and alignment of the FrpB hypervariable sequence regions. (A) Superposition of two hypervariable regions F5-1 purple, F3-3 (A chain) green. (B) Sequence alignment of the HR regions from the major FrpB variants.

    Techniques Used: Sequencing

    Structure and iron recognition by FrpB. (A) FrpB F3-3 variant trimer, viewed from above. (B) Detail of the Fe binding site in FrpB F5-1; Fe coordinating residues are marked. The anomalous difference map is shown in purple, contoured at 8σ, superimposed on the Fe atom. (C) Ribbon plot of FrpB F5-1, coloured by secondary structure, indicating the relative positions of Fe (arrowed), the helix-loop HR region (circled) and the outer membrane.
    Figure Legend Snippet: Structure and iron recognition by FrpB. (A) FrpB F3-3 variant trimer, viewed from above. (B) Detail of the Fe binding site in FrpB F5-1; Fe coordinating residues are marked. The anomalous difference map is shown in purple, contoured at 8σ, superimposed on the Fe atom. (C) Ribbon plot of FrpB F5-1, coloured by secondary structure, indicating the relative positions of Fe (arrowed), the helix-loop HR region (circled) and the outer membrane.

    Techniques Used: Variant Assay, Binding Assay

    Comparison of FrpB with heme transporter structures. Superposition of FrpB F5-1 (red), ShuA (PDB 3FHH; blue) and HasR (PDB 3DDR; magenta). ShuA and HasR were superimposed onto the FrpB F5-1 structure using the SSM superpose function within Coot [44] .
    Figure Legend Snippet: Comparison of FrpB with heme transporter structures. Superposition of FrpB F5-1 (red), ShuA (PDB 3FHH; blue) and HasR (PDB 3DDR; magenta). ShuA and HasR were superimposed onto the FrpB F5-1 structure using the SSM superpose function within Coot [44] .

    Techniques Used:

    13) Product Images from "ERG Induces Epigenetic Activation of Tudor Domain-Containing Protein 1 (TDRD1) in ERG Rearrangement-Positive Prostate Cancer"

    Article Title: ERG Induces Epigenetic Activation of Tudor Domain-Containing Protein 1 (TDRD1) in ERG Rearrangement-Positive Prostate Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0059976

    ERG-induced loss of epigenetic repression at the TDRD1 promoter is a major mechanism of TDRD1 overexpression. (A) DNA methylation analysis of the TDRD1 promoter-associated CpG island in prostate cell lines by bisulfite sequencing. Average methylation level of the whole CpG island calculated from five sequenced colonies is shown (%). (B) Analysis of mRNA expression in LNCaP cells after treatment with the demethylating agent 5-aza-2′-deoxycytidine. Insert: analysis of protein expression in LNCaP cells after treatment with 5-aza-2′-deoxycytidine. 75µg of protein lysate from LNCaP cells was used per lane. (C) Analysis of mRNA expression in stable LNCaP clones overexpressing ERG . Two independent experiments were performed in triplicate. Insert: ERG expression analysis at 48 h in LNCaP clones by western blotting. (D) Bisulfite sequencing of the TDRD1 promoter-associated CpG island in LNCaP cells 48 h after induction of ERG expression with doxycycline. (E) Bisulfite sequencing of the TDRD1 promoter-associated CpG island 96 h after silencing of ERG in VCaP cells. The data shown in (D) and (E) are mean % of methylation of the entire CpG island calculated from 11–12 sequenced clones.
    Figure Legend Snippet: ERG-induced loss of epigenetic repression at the TDRD1 promoter is a major mechanism of TDRD1 overexpression. (A) DNA methylation analysis of the TDRD1 promoter-associated CpG island in prostate cell lines by bisulfite sequencing. Average methylation level of the whole CpG island calculated from five sequenced colonies is shown (%). (B) Analysis of mRNA expression in LNCaP cells after treatment with the demethylating agent 5-aza-2′-deoxycytidine. Insert: analysis of protein expression in LNCaP cells after treatment with 5-aza-2′-deoxycytidine. 75µg of protein lysate from LNCaP cells was used per lane. (C) Analysis of mRNA expression in stable LNCaP clones overexpressing ERG . Two independent experiments were performed in triplicate. Insert: ERG expression analysis at 48 h in LNCaP clones by western blotting. (D) Bisulfite sequencing of the TDRD1 promoter-associated CpG island in LNCaP cells 48 h after induction of ERG expression with doxycycline. (E) Bisulfite sequencing of the TDRD1 promoter-associated CpG island 96 h after silencing of ERG in VCaP cells. The data shown in (D) and (E) are mean % of methylation of the entire CpG island calculated from 11–12 sequenced clones.

    Techniques Used: Over Expression, DNA Methylation Assay, Methylation Sequencing, Methylation, Expressing, Clone Assay, Western Blot

    TDRD1 does not control LINE1 activity in TMPRSS2:ERG -positive VCaP cells. (A) mRNA expression analysis of PIWIL genes in prostate cell lines by qRT-PCR. Testis RNA was used as a positive control. (B) mRNA expression analysis of LINE1 ORF2 in VCaP cells following 5 days of treatment with 5-aza-2′-deoxycytidine. (C) mRNA expression analysis of LINE1 ORF2 in VCaP cells following prolonged (8 days) ERG or TDRD1 silencing. (D) Metabolic viability assay of VCaP cells treated with siRNAs. One (B), two (A) or three (C, D) independent experiments were performed in triplicate.
    Figure Legend Snippet: TDRD1 does not control LINE1 activity in TMPRSS2:ERG -positive VCaP cells. (A) mRNA expression analysis of PIWIL genes in prostate cell lines by qRT-PCR. Testis RNA was used as a positive control. (B) mRNA expression analysis of LINE1 ORF2 in VCaP cells following 5 days of treatment with 5-aza-2′-deoxycytidine. (C) mRNA expression analysis of LINE1 ORF2 in VCaP cells following prolonged (8 days) ERG or TDRD1 silencing. (D) Metabolic viability assay of VCaP cells treated with siRNAs. One (B), two (A) or three (C, D) independent experiments were performed in triplicate.

    Techniques Used: Activity Assay, Expressing, Quantitative RT-PCR, Positive Control, Viability Assay

    14) Product Images from "Elongated Membrane Tethers, Individually Anchored by High Affinity ?4?1/VCAM-1 Complexes, Are the Quantal Units of Monocyte Arrests"

    Article Title: Elongated Membrane Tethers, Individually Anchored by High Affinity ?4?1/VCAM-1 Complexes, Are the Quantal Units of Monocyte Arrests

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064187

    AFM measurement of THP-1 adhesion to immobilized adhesion molecules. (A) AFM force-displacement curve of THP-1 adhesion to VCAM-1 co-immobilized with MCP-1. The approach (gray) and retract (black) traces of the AFM measurement are shown. Shaded area represents the work of detachment done by the cantilever to completely detach the cell from the substrate. Vertical arrows denote rupture events mediated by cytoskeleton-anchored (*) or membrane-tethered integrins (#). The inset is a representation of the experimental system. A flexible AFM cantilever was used to immobilize a THP-1 cell expressing α 4 β 1 and LFA-1 integrins and CCR-2 receptors of MCP-1, which was coimmobilized with ICAM-1 or VCAM-1 on the dish surface. Box-and-whisker plots of the (B) work of detachment, (C) detachment distance and detachment time, and (D) detachment force of THP-1 cell adhesion to VCAM-1 and ICAM-1, with and without co-immobilized MCP-1. The number in parenthesis above the plots corresponds to the number of cells studied. Numbers above each horizontal line represent the p-value from Mann-Whitney tests between respective measurement conditions. AFM measurements were acquired with a scan speed of ∼3 µm/s and a dwell time of 100 ms between approach and retract traces.
    Figure Legend Snippet: AFM measurement of THP-1 adhesion to immobilized adhesion molecules. (A) AFM force-displacement curve of THP-1 adhesion to VCAM-1 co-immobilized with MCP-1. The approach (gray) and retract (black) traces of the AFM measurement are shown. Shaded area represents the work of detachment done by the cantilever to completely detach the cell from the substrate. Vertical arrows denote rupture events mediated by cytoskeleton-anchored (*) or membrane-tethered integrins (#). The inset is a representation of the experimental system. A flexible AFM cantilever was used to immobilize a THP-1 cell expressing α 4 β 1 and LFA-1 integrins and CCR-2 receptors of MCP-1, which was coimmobilized with ICAM-1 or VCAM-1 on the dish surface. Box-and-whisker plots of the (B) work of detachment, (C) detachment distance and detachment time, and (D) detachment force of THP-1 cell adhesion to VCAM-1 and ICAM-1, with and without co-immobilized MCP-1. The number in parenthesis above the plots corresponds to the number of cells studied. Numbers above each horizontal line represent the p-value from Mann-Whitney tests between respective measurement conditions. AFM measurements were acquired with a scan speed of ∼3 µm/s and a dwell time of 100 ms between approach and retract traces.

    Techniques Used: Expressing, Whisker Assay, MANN-WHITNEY, Mass Spectrometry

    15) Product Images from "HDLs Protect Pancreatic ?-Cells Against ER Stress by Restoring Protein Folding and Trafficking"

    Article Title: HDLs Protect Pancreatic ?-Cells Against ER Stress by Restoring Protein Folding and Trafficking

    Journal: Diabetes

    doi: 10.2337/db11-1221

    HDLs restore ER to Golgi trafficking in palmitate (Palm)-treated cells. MIN6 cells were infected with VSVG-GFP-encoding lentiviruses and treated or not with 0.4 mmol/L palmitate in the presence or in the absence of 1 mmol/L HDLs for 48 h (in each case, BSA was present at a 0.3% concentration). The cells were then incubated 5 h at 40°C. They were then treated for 15 min with 5 μmol/L cycloheximide before switching the temperature to 32°C for 0 or 30 min (note that the temperature shifts and/or cycloheximide did not induce apoptosis; see Supplementary Fig. 8 ). The cells were stained with an antibody recognizing the Golgi marker GM130 (red staining; left part of A ) or processed as described in Fig. 5 A and B (right part of A ). Nuclei were stained in blue with the Hoechst 33342 dye. Representative examples of the different locations of VSVG in cells are shown in B (VSVG mainly in the ER when the GFP signal does not colocalize with GM130 staining; VSVG mainly in the Golgi when these two signals extensively colocalize; and intermediate situation when the GFP signal only partially colocalizes with the Golgi marker). C : the quantitation of the percentage of cells with VSVG mainly localized in the Golgi, and D depicts the quantitation of the percentage of cells with correctly folded VSVG (results derived from 5 independent experiments each). *Significant differences. Ctrl, control. (A high-quality digital representation of this figure is available in the online issue.)
    Figure Legend Snippet: HDLs restore ER to Golgi trafficking in palmitate (Palm)-treated cells. MIN6 cells were infected with VSVG-GFP-encoding lentiviruses and treated or not with 0.4 mmol/L palmitate in the presence or in the absence of 1 mmol/L HDLs for 48 h (in each case, BSA was present at a 0.3% concentration). The cells were then incubated 5 h at 40°C. They were then treated for 15 min with 5 μmol/L cycloheximide before switching the temperature to 32°C for 0 or 30 min (note that the temperature shifts and/or cycloheximide did not induce apoptosis; see Supplementary Fig. 8 ). The cells were stained with an antibody recognizing the Golgi marker GM130 (red staining; left part of A ) or processed as described in Fig. 5 A and B (right part of A ). Nuclei were stained in blue with the Hoechst 33342 dye. Representative examples of the different locations of VSVG in cells are shown in B (VSVG mainly in the ER when the GFP signal does not colocalize with GM130 staining; VSVG mainly in the Golgi when these two signals extensively colocalize; and intermediate situation when the GFP signal only partially colocalizes with the Golgi marker). C : the quantitation of the percentage of cells with VSVG mainly localized in the Golgi, and D depicts the quantitation of the percentage of cells with correctly folded VSVG (results derived from 5 independent experiments each). *Significant differences. Ctrl, control. (A high-quality digital representation of this figure is available in the online issue.)

    Techniques Used: Infection, Concentration Assay, Incubation, Staining, Marker, Quantitation Assay, Derivative Assay

    HDL-mediated β-cell protection against TG-induced apoptosis is inhibited by BFA. A and B : MIN6 cells were infected with VSVG-GFP-encoding lentiviruses and treated 2 days later with or without 0.5 μmol/L TG in the presence or in the absence of 1 mmol/L HDLs for 5 h at 40°C. The cells were then incubated or not for an additional 1-h time period at 32°C. The presence of folded VSVG was assessed by immunocytochemistry on permeabilized cells using an antibody specifically recognizing the correctly folded form of the protein. The percentage of cells expressing folded VSVG was quantitated and shown in B . C – E : Cells were treated as in A except that nonpermeabilized cells were labeled with an antibody directed against the ectopic part of VSVG. The percentage of cells expressing VSVG at the cell surface was quantitated and shown in D . Quantification of the GFP fluorescence intensity in VSVG-GFP expressing cells is presented in E . F and G : MIN6 cells infected with VSVG-GFP-encoding lentiviruses were preincubated or not with 250 ng/mL BFA for 2 h before being subjected to the indicated combinations of 0.5 μmol/L TG and 1 mmol/L HDLs for an additional 22-h period. Permeabilized cells were then stained with an antibody recognizing GM130, a specific Golgi marker ( F ). Alternatively, apoptosis was assessed by scoring cells with pycnotic and/or fragmented nucleus ( G ). Means with different symbol (# or ) are significantly different. Nuclei were stained in blue with the Hoechst 33342 dye. *Significant differences. NS, no significant differences. VEH, vehicle; C, control. (A high-quality digital representation of this figure is available in the online issue.)
    Figure Legend Snippet: HDL-mediated β-cell protection against TG-induced apoptosis is inhibited by BFA. A and B : MIN6 cells were infected with VSVG-GFP-encoding lentiviruses and treated 2 days later with or without 0.5 μmol/L TG in the presence or in the absence of 1 mmol/L HDLs for 5 h at 40°C. The cells were then incubated or not for an additional 1-h time period at 32°C. The presence of folded VSVG was assessed by immunocytochemistry on permeabilized cells using an antibody specifically recognizing the correctly folded form of the protein. The percentage of cells expressing folded VSVG was quantitated and shown in B . C – E : Cells were treated as in A except that nonpermeabilized cells were labeled with an antibody directed against the ectopic part of VSVG. The percentage of cells expressing VSVG at the cell surface was quantitated and shown in D . Quantification of the GFP fluorescence intensity in VSVG-GFP expressing cells is presented in E . F and G : MIN6 cells infected with VSVG-GFP-encoding lentiviruses were preincubated or not with 250 ng/mL BFA for 2 h before being subjected to the indicated combinations of 0.5 μmol/L TG and 1 mmol/L HDLs for an additional 22-h period. Permeabilized cells were then stained with an antibody recognizing GM130, a specific Golgi marker ( F ). Alternatively, apoptosis was assessed by scoring cells with pycnotic and/or fragmented nucleus ( G ). Means with different symbol (# or ) are significantly different. Nuclei were stained in blue with the Hoechst 33342 dye. *Significant differences. NS, no significant differences. VEH, vehicle; C, control. (A high-quality digital representation of this figure is available in the online issue.)

    Techniques Used: Infection, Incubation, Immunocytochemistry, Expressing, Labeling, Fluorescence, Staining, Marker

    HDLs inhibit the induction of stress markers by TG and palmitate. MIN6 cells and rat islets were left untreated (control [C]) or treated with 0.5 μmol/L TG during 6 h ( A – C and E ) or 24 h ( D and F ) in the presence or in the absence of 1 mmol/L HDLs. Alternatively, the cells were treated with 0.3% BSA (BSA) or with 0.3% BSA/0.4 mmol/L palmitate (P) in the presence or in the absence of 1 mmol/L HDLs for 24 h ( A and B ) or 48 h ( C ). The cells were then lysed, and RNA and proteins were isolated. The extent of XBP1 mRNA splicing was then determined ( A ). The pound sign (#) in A indicates an unspecific band (see research design and methods ). CHOP mRNA expression was determined by quantitative PCR ( B ). Western blot analysis were performed to assess protein expression of CHOP ( C ), BiP ( D ), phospho-PERK ( E ), and phospho- and total JNK ( F ). The experiments presented in E and F were repeated once and twice, respectively, and yielded similar results. *Significant differences. VEH, vehicle.
    Figure Legend Snippet: HDLs inhibit the induction of stress markers by TG and palmitate. MIN6 cells and rat islets were left untreated (control [C]) or treated with 0.5 μmol/L TG during 6 h ( A – C and E ) or 24 h ( D and F ) in the presence or in the absence of 1 mmol/L HDLs. Alternatively, the cells were treated with 0.3% BSA (BSA) or with 0.3% BSA/0.4 mmol/L palmitate (P) in the presence or in the absence of 1 mmol/L HDLs for 24 h ( A and B ) or 48 h ( C ). The cells were then lysed, and RNA and proteins were isolated. The extent of XBP1 mRNA splicing was then determined ( A ). The pound sign (#) in A indicates an unspecific band (see research design and methods ). CHOP mRNA expression was determined by quantitative PCR ( B ). Western blot analysis were performed to assess protein expression of CHOP ( C ), BiP ( D ), phospho-PERK ( E ), and phospho- and total JNK ( F ). The experiments presented in E and F were repeated once and twice, respectively, and yielded similar results. *Significant differences. VEH, vehicle.

    Techniques Used: Isolation, Expressing, Real-time Polymerase Chain Reaction, Western Blot

    HDLs protect β-cells against apoptosis induced by ER stress. A : Human islets from cadaveric donors were dissociated using trypsin and plated. The next day, islets were treated (TG) or not (control [C]) with 10 μmol/L TG in the presence (HDL) or in the absence (vehicle [VEH]) of HDLs for 24 h. Cells were then fixed, and apoptosis was assessed by scoring pycnotic nucleus. B : Cultured rat islets were incubated 24 h in serum-free RPMI 1640 medium containing 5 g/L BSA and 10 mmol/L glucose with the indicated combinations of 1 μmol/L TG and 1 mmol/L HDLs. Cell death was determined by transferase-mediated dUTP nick-end labeling (TUNEL) in insulin-expressing cells on histological sections of the islets. Results are expressed as the percentage of apoptotic cells among insulin-positive cells in a given islet section. A minimum of 2,000 cells from at least 20 islets have been scored from two independent experiments. C : MIN6 cells were treated with 0.5 μmol/L TG in the presence or in the absence of 1 mmol/L HDLs for 24 h. Cells were then fixed, and apoptosis was determined. Alternatively, the cells were lysed and the extent of caspase-3 activation was assessed by Western blotting using an antibody recognizing the cleaved active form of the protease. An actin-specific antibody was also used on the same blot to assess the evenness of loading. D : MIN6 cells were left untreated (control [Ctrl]) or treated for 48 h with 0.3% BSA (BSA) or 0.3% BSA/0.4 mmol/L palmitate (P) in the presence or in the absence of 1 mmol/L HDLs. Apoptosis was then scored as in A . E and F : Cultured rat islets were incubated for a week in serum-free RPMI 1640 medium containing 5 g/L BSA and 10 or 30 mmol/L glucose (labeled G10 and G30 in the figure) in the presence (HDL) or in the absence (vehicle [VEH]) of 1 mmol/L HDLs. Apoptosis was then assessed as in B . E : representative examples of TUNEL staining (green staining) in insulin-positive cells (red staining; nuclei are stained in blue with DAPI). The corresponding quantitation is shown in F . *Significant differences. (A high-quality digital representation of this figure is available in the online issue.)
    Figure Legend Snippet: HDLs protect β-cells against apoptosis induced by ER stress. A : Human islets from cadaveric donors were dissociated using trypsin and plated. The next day, islets were treated (TG) or not (control [C]) with 10 μmol/L TG in the presence (HDL) or in the absence (vehicle [VEH]) of HDLs for 24 h. Cells were then fixed, and apoptosis was assessed by scoring pycnotic nucleus. B : Cultured rat islets were incubated 24 h in serum-free RPMI 1640 medium containing 5 g/L BSA and 10 mmol/L glucose with the indicated combinations of 1 μmol/L TG and 1 mmol/L HDLs. Cell death was determined by transferase-mediated dUTP nick-end labeling (TUNEL) in insulin-expressing cells on histological sections of the islets. Results are expressed as the percentage of apoptotic cells among insulin-positive cells in a given islet section. A minimum of 2,000 cells from at least 20 islets have been scored from two independent experiments. C : MIN6 cells were treated with 0.5 μmol/L TG in the presence or in the absence of 1 mmol/L HDLs for 24 h. Cells were then fixed, and apoptosis was determined. Alternatively, the cells were lysed and the extent of caspase-3 activation was assessed by Western blotting using an antibody recognizing the cleaved active form of the protease. An actin-specific antibody was also used on the same blot to assess the evenness of loading. D : MIN6 cells were left untreated (control [Ctrl]) or treated for 48 h with 0.3% BSA (BSA) or 0.3% BSA/0.4 mmol/L palmitate (P) in the presence or in the absence of 1 mmol/L HDLs. Apoptosis was then scored as in A . E and F : Cultured rat islets were incubated for a week in serum-free RPMI 1640 medium containing 5 g/L BSA and 10 or 30 mmol/L glucose (labeled G10 and G30 in the figure) in the presence (HDL) or in the absence (vehicle [VEH]) of 1 mmol/L HDLs. Apoptosis was then assessed as in B . E : representative examples of TUNEL staining (green staining) in insulin-positive cells (red staining; nuclei are stained in blue with DAPI). The corresponding quantitation is shown in F . *Significant differences. (A high-quality digital representation of this figure is available in the online issue.)

    Techniques Used: Cell Culture, Incubation, End Labeling, TUNEL Assay, Expressing, Activation Assay, Western Blot, Labeling, Staining, Quantitation Assay

    HDLs prevent TG- and palmitate-induced ER morphology alterations. MIN6 cells were plated on glass slides previously coated with poly- l -lysine and incubated for 6 h with the indicated treatments ( A ). Alternatively, the cells were treated for 6 h with TG and then incubated or not with HDLs for an additional 18 h-period ( B ). C : cells were incubated for 12 h with 0.3% BSA, 0.4 mmol/L palmitate, and 1 mmol/L HDLs in the indicated combinations. The cells were then processed for electron microscopy as described in research design and methods . Ctrl, control.
    Figure Legend Snippet: HDLs prevent TG- and palmitate-induced ER morphology alterations. MIN6 cells were plated on glass slides previously coated with poly- l -lysine and incubated for 6 h with the indicated treatments ( A ). Alternatively, the cells were treated for 6 h with TG and then incubated or not with HDLs for an additional 18 h-period ( B ). C : cells were incubated for 12 h with 0.3% BSA, 0.4 mmol/L palmitate, and 1 mmol/L HDLs in the indicated combinations. The cells were then processed for electron microscopy as described in research design and methods . Ctrl, control.

    Techniques Used: Incubation, Electron Microscopy

    Insulin overexpression-induced β-cell apoptosis is inhibited by HDLs. MIN6 cells were infected with lentiviruses encoding the indicated constructs. Three days later, cells were trypsinized and plated in new culture dishes for 4 days, the last two days in the presence or in the absence of 1 mmol/L HDLs. Apoptosis was then determined by scoring pycnotic and fragmented nuclei ( A ). Alternatively, 24 h after the infection, the extent of XBP1 mRNA splicing ( B ) and CHOP mRNA expression ( C ) were determined. *indicates significant differences. VEH, vehicle; NS, no significant differences.
    Figure Legend Snippet: Insulin overexpression-induced β-cell apoptosis is inhibited by HDLs. MIN6 cells were infected with lentiviruses encoding the indicated constructs. Three days later, cells were trypsinized and plated in new culture dishes for 4 days, the last two days in the presence or in the absence of 1 mmol/L HDLs. Apoptosis was then determined by scoring pycnotic and fragmented nuclei ( A ). Alternatively, 24 h after the infection, the extent of XBP1 mRNA splicing ( B ) and CHOP mRNA expression ( C ) were determined. *indicates significant differences. VEH, vehicle; NS, no significant differences.

    Techniques Used: Over Expression, Infection, Construct, Expressing

    Oxidized HDLs (oxHDL) do not protect β-cells against TG-induced apoptosis. MIN6 cells were treated with 0.5 μmol/L TG in the presence or in the absence of 1 mmol/L nonoxidized or oxidized HDLs (oxidation performed during 8 or 16 h) for 24 h. Cells were then fixed, and apoptosis was assessed by scoring pycnotic nuclei. *Significant differences. VEH, vehicle; NS, no significant differences; C, control.
    Figure Legend Snippet: Oxidized HDLs (oxHDL) do not protect β-cells against TG-induced apoptosis. MIN6 cells were treated with 0.5 μmol/L TG in the presence or in the absence of 1 mmol/L nonoxidized or oxidized HDLs (oxidation performed during 8 or 16 h) for 24 h. Cells were then fixed, and apoptosis was assessed by scoring pycnotic nuclei. *Significant differences. VEH, vehicle; NS, no significant differences; C, control.

    Techniques Used:

    16) Product Images from "Cross-species genomic and epigenomic landscape of retinoblastoma"

    Article Title: Cross-species genomic and epigenomic landscape of retinoblastoma

    Journal: Oncotarget

    doi:

    Analysis of dsDNA-Break Repair in Retinoblastoma (A) Comet analysis of retinoblastoma cell lines, human retinoblastoma xenografts, and primary mouse retinoblastoma tumors. The number of cells analyzed in each experiment is indicated at the top of the plot. TERT-immortalized human fibroblasts (BJ) were used as the normal human negative-control; MEF cells were used as the normal mouse negative-control, and Brca;p53 -deficient medulloblastoma cells were used as the positive control. (B-G) Cells were exposed to 10 Gy irradiation (IR), and dsDNA-break repair of BJ and SJRB001X tumor cells (C, D), MEF, RbTKO and p53TKO tumor cells (E-G) was monitored by performing comet assays at different time points. (B) Representative images of nuclei stained with Sytox green before and after exposure to 10 Gy IR. (H) Immunostaining of primary human retinoblastoma to detect TP53BP1 foci (arrow). (I, J) Immunofluorescence detection of TP53BP1 (red) in DAPI-stained nuclei (blue) of untreated BJ and SJRB001X cells and after exposure to 5 Gy IR. Foci of TP53BP1 at sites of dsDNA breaks are highlighted with arrows. Histogram of the proportion of cells with TP53BP1 foci before and after exposure to 5 Gy IR in (K) human retinoblastoma cell lines (Weri1 and Y79), (L) two independent retinoblastoma orthotopic xenografts (SJRB001X and SJRB002X), (M) mouse retinoblastoma cell line (SJmRBL8), and (N) the three primary mouse retinoblastoma (RBTKO, MDMX, p53TKO). p values compared to untreated samples.
    Figure Legend Snippet: Analysis of dsDNA-Break Repair in Retinoblastoma (A) Comet analysis of retinoblastoma cell lines, human retinoblastoma xenografts, and primary mouse retinoblastoma tumors. The number of cells analyzed in each experiment is indicated at the top of the plot. TERT-immortalized human fibroblasts (BJ) were used as the normal human negative-control; MEF cells were used as the normal mouse negative-control, and Brca;p53 -deficient medulloblastoma cells were used as the positive control. (B-G) Cells were exposed to 10 Gy irradiation (IR), and dsDNA-break repair of BJ and SJRB001X tumor cells (C, D), MEF, RbTKO and p53TKO tumor cells (E-G) was monitored by performing comet assays at different time points. (B) Representative images of nuclei stained with Sytox green before and after exposure to 10 Gy IR. (H) Immunostaining of primary human retinoblastoma to detect TP53BP1 foci (arrow). (I, J) Immunofluorescence detection of TP53BP1 (red) in DAPI-stained nuclei (blue) of untreated BJ and SJRB001X cells and after exposure to 5 Gy IR. Foci of TP53BP1 at sites of dsDNA breaks are highlighted with arrows. Histogram of the proportion of cells with TP53BP1 foci before and after exposure to 5 Gy IR in (K) human retinoblastoma cell lines (Weri1 and Y79), (L) two independent retinoblastoma orthotopic xenografts (SJRB001X and SJRB002X), (M) mouse retinoblastoma cell line (SJmRBL8), and (N) the three primary mouse retinoblastoma (RBTKO, MDMX, p53TKO). p values compared to untreated samples.

    Techniques Used: Negative Control, Positive Control, Irradiation, Staining, Immunostaining, Immunofluorescence

    17) Product Images from "Epigallocatechin gallate attenuates L-DOPA-induced apoptosis in rat PC12 cells"

    Article Title: Epigallocatechin gallate attenuates L-DOPA-induced apoptosis in rat PC12 cells

    Journal: Nutrition Research and Practice

    doi: 10.4162/nrp.2013.7.4.249

    Flow cytometric histograms of PC 12 cells after exposure to 150 µM L-DOPA alone for 24 h or preincubated with 100 µM EGCG for 30 min before treatment with L-DOPA. (A) After incubation, cells were harvested and stained with propidium iodide. Relative DNA content was analyzed by flow cytometry. The X -axis represents DNA content and the Y -axis represents the number of cells. (B) The results shown in present the mean ± SEM of six experiments. * P
    Figure Legend Snippet: Flow cytometric histograms of PC 12 cells after exposure to 150 µM L-DOPA alone for 24 h or preincubated with 100 µM EGCG for 30 min before treatment with L-DOPA. (A) After incubation, cells were harvested and stained with propidium iodide. Relative DNA content was analyzed by flow cytometry. The X -axis represents DNA content and the Y -axis represents the number of cells. (B) The results shown in present the mean ± SEM of six experiments. * P

    Techniques Used: Flow Cytometry, Incubation, Staining, Cytometry

    18) Product Images from "Schwann-like cells seeded in acellular nerve grafts improve nerve regeneration"

    Article Title: Schwann-like cells seeded in acellular nerve grafts improve nerve regeneration

    Journal: BMC Musculoskeletal Disorders

    doi: 10.1186/1471-2474-15-165

    S-100 and VEGF immunostaining and IOD value analyses at 12 weeks postoperatively. S-100 immunostaining ( A-C , scale bar = 10 μm) and VEGF immunostaining ( D-F , scale bar = 20 μm) at 12 weeks postoperatively with SLCs (A and D) , BM-MSCs (B and E) and isografts (C and F) . The IOD values of the positive expression of S-100 and VEGF in the regenerated nerves (G) . The isograft and ANG + SLC groups showed a significantly higher level in both S-100 and VEGF immunostaining compared with the ANG + BM-MSC group. The data were shown as the mean ± SEM. * P
    Figure Legend Snippet: S-100 and VEGF immunostaining and IOD value analyses at 12 weeks postoperatively. S-100 immunostaining ( A-C , scale bar = 10 μm) and VEGF immunostaining ( D-F , scale bar = 20 μm) at 12 weeks postoperatively with SLCs (A and D) , BM-MSCs (B and E) and isografts (C and F) . The IOD values of the positive expression of S-100 and VEGF in the regenerated nerves (G) . The isograft and ANG + SLC groups showed a significantly higher level in both S-100 and VEGF immunostaining compared with the ANG + BM-MSC group. The data were shown as the mean ± SEM. * P

    Techniques Used: Immunostaining, Expressing

    19) Product Images from "Comparison of the neuropoietic activity of gene-modified versus parental mesenchymal stromal cells and the identification of soluble and extracellular matrix-related neuropoietic mediators"

    Article Title: Comparison of the neuropoietic activity of gene-modified versus parental mesenchymal stromal cells and the identification of soluble and extracellular matrix-related neuropoietic mediators

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/scrt418

    Role of human growth factors in induction of rat neural markers in various culture settings; qRT-PCR. (A) Effect of FGF2 inhibition on rat nestin. Rat neural cells were stimulated with SB623 cell-derived conditioned medium (25%), which increased the nestin expression. The presence of FGF2-neutralizing antibody (bFM1), but not FGF2-specific nonneutralizing antibody (bMF2), concentration-dependently inhibited the nestin increase. (B) Effect of human FGF1 inhibition on rat nestin. Rat neural cells were stimulated by MSCs (60 or 200 cells/well), MSC-CM (5%), or recombinant FGF1, or FGF2 (both at 5 ng/ml), which led to the induction of rat nestin. The application of the anti-human FGF1 neutralizing antibody decreased nestin mRNA levels induced by all stimuli but the recombinant FGF2. (C) Effect of BMP inhibition on rat GFAP induction. Rat GFAP was induced by coculturing rat neural cells with SB623 (200 cells/well). A BMP inhibitor noggin concentration-dependently decreased the GFAP induction. (D) Effect of HGF silencing in MSC on rat CNP induction. Rat CNP expression was stimulated by coculturing rat neural cells with MSC transfected with either HGF siRNA (siHGF) or control siRNA (siControl). A lower CNP induction was observed in cocultures with siHGF-transfectants.
    Figure Legend Snippet: Role of human growth factors in induction of rat neural markers in various culture settings; qRT-PCR. (A) Effect of FGF2 inhibition on rat nestin. Rat neural cells were stimulated with SB623 cell-derived conditioned medium (25%), which increased the nestin expression. The presence of FGF2-neutralizing antibody (bFM1), but not FGF2-specific nonneutralizing antibody (bMF2), concentration-dependently inhibited the nestin increase. (B) Effect of human FGF1 inhibition on rat nestin. Rat neural cells were stimulated by MSCs (60 or 200 cells/well), MSC-CM (5%), or recombinant FGF1, or FGF2 (both at 5 ng/ml), which led to the induction of rat nestin. The application of the anti-human FGF1 neutralizing antibody decreased nestin mRNA levels induced by all stimuli but the recombinant FGF2. (C) Effect of BMP inhibition on rat GFAP induction. Rat GFAP was induced by coculturing rat neural cells with SB623 (200 cells/well). A BMP inhibitor noggin concentration-dependently decreased the GFAP induction. (D) Effect of HGF silencing in MSC on rat CNP induction. Rat CNP expression was stimulated by coculturing rat neural cells with MSC transfected with either HGF siRNA (siHGF) or control siRNA (siControl). A lower CNP induction was observed in cocultures with siHGF-transfectants.

    Techniques Used: Quantitative RT-PCR, Inhibition, Derivative Assay, Expressing, Concentration Assay, Recombinant, Transfection

    Comparison of expression and activity of TGM2 in SB623 and MSC; its functional analysis in ECM using siRNA. (A) Expression levels of TGM2 normalized to GAP were determined by using qRT-PCR in SB623/MSC pairs from several donors. Levels in SB623 cells were expressed relative to levels in parental MSCs, which were set on 1. (B) TGM2-crosslinking activity was measured by amounts of biotinylated cadaverine incorporated into PLL in the presence of SB623 or MSC cell lysates. The activity was then normalized to the total protein, and expressed compared with the parental MSCs, where the values were set on 1. (C) TGM2 was detected in ECM of MSCs and SB623 by using immunoblotting. The TGM2 signal was quantified densitometrically and normalized to the total ECM protein per lane. The total ECM protein was assessed by using duplicated gel: the gel was stained for protein; photographed; and the density of corresponding lane minus background determined. (The whole blot and gel are shown in Additional file 6 : Figure S5). (D) Nestin expression was quantified by using qRT-PCR in rat neural cells grown on ECM produced by SB623 transfected with either siTGM2 or siControl. Nestin levels on siControl-ECM were set on 1. The graph represents means from three experiments; error bar represents standard error of mean; * P
    Figure Legend Snippet: Comparison of expression and activity of TGM2 in SB623 and MSC; its functional analysis in ECM using siRNA. (A) Expression levels of TGM2 normalized to GAP were determined by using qRT-PCR in SB623/MSC pairs from several donors. Levels in SB623 cells were expressed relative to levels in parental MSCs, which were set on 1. (B) TGM2-crosslinking activity was measured by amounts of biotinylated cadaverine incorporated into PLL in the presence of SB623 or MSC cell lysates. The activity was then normalized to the total protein, and expressed compared with the parental MSCs, where the values were set on 1. (C) TGM2 was detected in ECM of MSCs and SB623 by using immunoblotting. The TGM2 signal was quantified densitometrically and normalized to the total ECM protein per lane. The total ECM protein was assessed by using duplicated gel: the gel was stained for protein; photographed; and the density of corresponding lane minus background determined. (The whole blot and gel are shown in Additional file 6 : Figure S5). (D) Nestin expression was quantified by using qRT-PCR in rat neural cells grown on ECM produced by SB623 transfected with either siTGM2 or siControl. Nestin levels on siControl-ECM were set on 1. The graph represents means from three experiments; error bar represents standard error of mean; * P

    Techniques Used: Expressing, Activity Assay, Functional Assay, Quantitative RT-PCR, Staining, Produced, Transfection

    Comparison of neuropoietic activity of SB623 and MSCs in cocultures with rat embryonic neural cells. (A) Rat neural cells were grown in the presence or absence of MSCs or SB623 (rat/human cell ratio was 10:1) and immunostained for rat nestin (upper panel) and GFAP (middle panel) on day 5, or for CNP on day 12. Nuclei were stained with DAPI (B) An example of microplate neuropoiesis assay data: a comparison of rat neural differentiation marker induction in cocultures of rat cells with MSCs and SB623 from Donor A. Rat neural cells (5,000/well) were cocultured with 500, 250, and 125 cells/well of either MSCs or SB623; and expression of rat-specific nestin, GFAP, and CNP, and human-specific GAP was quantified by using qRT-PCR. Stimulation with 10% MSC-CM and no stimulation (“No add”) were used as positive control and background, respectively. Relative units correspond to standard samples used in qPCR run. Error bars represent SD of biologic duplicates. (C) Neuropoietic coefficients of MSCs and SB623 from Donor A were calculated based on data presented in (B) by first subtracting the background expression of a corresponding neural marker and then normalizing the expression of the neural marker to the human GAP for each number of human cells per well, followed by averaging normalized values. Error bars represent SD from three normalized values.
    Figure Legend Snippet: Comparison of neuropoietic activity of SB623 and MSCs in cocultures with rat embryonic neural cells. (A) Rat neural cells were grown in the presence or absence of MSCs or SB623 (rat/human cell ratio was 10:1) and immunostained for rat nestin (upper panel) and GFAP (middle panel) on day 5, or for CNP on day 12. Nuclei were stained with DAPI (B) An example of microplate neuropoiesis assay data: a comparison of rat neural differentiation marker induction in cocultures of rat cells with MSCs and SB623 from Donor A. Rat neural cells (5,000/well) were cocultured with 500, 250, and 125 cells/well of either MSCs or SB623; and expression of rat-specific nestin, GFAP, and CNP, and human-specific GAP was quantified by using qRT-PCR. Stimulation with 10% MSC-CM and no stimulation (“No add”) were used as positive control and background, respectively. Relative units correspond to standard samples used in qPCR run. Error bars represent SD of biologic duplicates. (C) Neuropoietic coefficients of MSCs and SB623 from Donor A were calculated based on data presented in (B) by first subtracting the background expression of a corresponding neural marker and then normalizing the expression of the neural marker to the human GAP for each number of human cells per well, followed by averaging normalized values. Error bars represent SD from three normalized values.

    Techniques Used: Activity Assay, Staining, Marker, Expressing, Quantitative RT-PCR, Positive Control, Real-time Polymerase Chain Reaction

    Comparison of presynaptic puncta formation in cocultures of rat embryonic neural cells with MSCs or SB623; immunostaining. (A) Immunostaining for VGLUT (day 7) and VGAT (day 11); rat/human cell ratio, 50:1. Bar, 50 μm. (B) Quantification of VGAT-immunoreactive puncta per neurite length (averaged from 10 microscopic fields, one to four neurites/field), day 11. (C) Typical distribution and size of VGAT-positive puncta in axonal processes in cocultures with MSCs (upper) or SB623 (lower), day 13. Neurites are outlined manually. Bar, 50 um.
    Figure Legend Snippet: Comparison of presynaptic puncta formation in cocultures of rat embryonic neural cells with MSCs or SB623; immunostaining. (A) Immunostaining for VGLUT (day 7) and VGAT (day 11); rat/human cell ratio, 50:1. Bar, 50 μm. (B) Quantification of VGAT-immunoreactive puncta per neurite length (averaged from 10 microscopic fields, one to four neurites/field), day 11. (C) Typical distribution and size of VGAT-positive puncta in axonal processes in cocultures with MSCs (upper) or SB623 (lower), day 13. Neurites are outlined manually. Bar, 50 um.

    Techniques Used: Immunostaining

    Comparison of SB623- and MSC-derived ECM in supporting nestin-positive cell growth. (A) Rat neural cells were grown for 5 days on MSC- or SB623-derived ECM and then immunostained for nestin (upper panel) and counterstained for nuclei (lower panel); magnification 100×. (B) Nestin-positive cells grown and stained as described in (A) were counted, and their numbers expressed as percentage of total nuclei. (C) Rat nestin expression in cells growing on MSC- or SB623-ECM was quantified by using qRT-PCR and normalized by the LDH activity released from either MSC- or SB623-ECM-producing cells during ECM preparation, correspondingly, to account for possible differences in cell numbers.
    Figure Legend Snippet: Comparison of SB623- and MSC-derived ECM in supporting nestin-positive cell growth. (A) Rat neural cells were grown for 5 days on MSC- or SB623-derived ECM and then immunostained for nestin (upper panel) and counterstained for nuclei (lower panel); magnification 100×. (B) Nestin-positive cells grown and stained as described in (A) were counted, and their numbers expressed as percentage of total nuclei. (C) Rat nestin expression in cells growing on MSC- or SB623-ECM was quantified by using qRT-PCR and normalized by the LDH activity released from either MSC- or SB623-ECM-producing cells during ECM preparation, correspondingly, to account for possible differences in cell numbers.

    Techniques Used: Derivative Assay, Staining, Expressing, Quantitative RT-PCR, Activity Assay

    20) Product Images from "Detection and quantification of lupus anticoagulants in plasma from heparin treated patients, using addition of polybrene"

    Article Title: Detection and quantification of lupus anticoagulants in plasma from heparin treated patients, using addition of polybrene

    Journal: Thrombosis Journal

    doi: 10.1186/1477-9560-4-3

    Effect of heparin neutralisers on the APTT of heparinised plasma . a) APTT values of Na-citrate plasma with heparin added in vitro to a final concentration of 0.5 U/ml. APTT reagent with cephalin 1/100 as phospholipid source. APTT in this heparinised plasma was unmeasurable without heparin neutraliser, i.e. clotting times > 120 sec. The figure shows the clotting times when increasing concentrations of the different heparin neutralisers were added. b) APTT values of Na-citrate plasma with heparin added to a final concentration of 0.5 U/ml. APTT reagent with cephalin 1/3200 as phospholipid source. Again, APTT was > 120 sec. when no heparin neutraliser were added. The figure shows the clotting times when increasing concentrations of the different heparin neutralisers were added.
    Figure Legend Snippet: Effect of heparin neutralisers on the APTT of heparinised plasma . a) APTT values of Na-citrate plasma with heparin added in vitro to a final concentration of 0.5 U/ml. APTT reagent with cephalin 1/100 as phospholipid source. APTT in this heparinised plasma was unmeasurable without heparin neutraliser, i.e. clotting times > 120 sec. The figure shows the clotting times when increasing concentrations of the different heparin neutralisers were added. b) APTT values of Na-citrate plasma with heparin added to a final concentration of 0.5 U/ml. APTT reagent with cephalin 1/3200 as phospholipid source. Again, APTT was > 120 sec. when no heparin neutraliser were added. The figure shows the clotting times when increasing concentrations of the different heparin neutralisers were added.

    Techniques Used: In Vitro, Concentration Assay, Coagulation, Size-exclusion Chromatography

    21) Product Images from "Cyclosporin A increases recovery after spinal cord injury but does not improve myelination by oligodendrocyte progenitor cell transplantation"

    Article Title: Cyclosporin A increases recovery after spinal cord injury but does not improve myelination by oligodendrocyte progenitor cell transplantation

    Journal: BMC Neuroscience

    doi: 10.1186/1471-2202-11-127

    Identification and differentiation of GFP-OPCs . The GFP-OPCs induced from spinal cord-derived NPCs were cultured in different media for 5 days. (A~C): In the basal-OPC-medium containing PDGF and bFGF, the cells display bipolar or tri-polar morphology, the typical morphology of OPC (A), more than 95% of cells express both A2B5 (B) and PDGFR (C). Inserts show higher power photographs of OPCs. (D~I) In the medium containing T3 and without PDGF and bFGF, the cells display a multipolar morphology (D, G), more than 95% of the cells express RIP (E, F), and almost no cells express GFAP (H, I).(J~O) In the medium containing 10%FBS without PDGF and bFGF, the cells display the typical process-bearing morphology of astrocytes (J, M), few cells express RIP (K, L) and nearly all cells express GFAP (N, O). Cells in B, C, E, F, H, I, K, L, N and O were counterstained with Hoechst33342 (blue), a nuclear dye. Scale bars: 25 μm.
    Figure Legend Snippet: Identification and differentiation of GFP-OPCs . The GFP-OPCs induced from spinal cord-derived NPCs were cultured in different media for 5 days. (A~C): In the basal-OPC-medium containing PDGF and bFGF, the cells display bipolar or tri-polar morphology, the typical morphology of OPC (A), more than 95% of cells express both A2B5 (B) and PDGFR (C). Inserts show higher power photographs of OPCs. (D~I) In the medium containing T3 and without PDGF and bFGF, the cells display a multipolar morphology (D, G), more than 95% of the cells express RIP (E, F), and almost no cells express GFAP (H, I).(J~O) In the medium containing 10%FBS without PDGF and bFGF, the cells display the typical process-bearing morphology of astrocytes (J, M), few cells express RIP (K, L) and nearly all cells express GFAP (N, O). Cells in B, C, E, F, H, I, K, L, N and O were counterstained with Hoechst33342 (blue), a nuclear dye. Scale bars: 25 μm.

    Techniques Used: Derivative Assay, Cell Culture

    22) Product Images from "?1-6 branching of cell surface glycoproteins may contribute to uveal melanoma progression by up-regulating cell motility"

    Article Title: ?1-6 branching of cell surface glycoproteins may contribute to uveal melanoma progression by up-regulating cell motility

    Journal: Molecular Vision

    doi:

    Effect of phaseolus vulgaris agglutinin on repair of wounds in monolayers of 92–1, Mel202, FM55P, and IGR-39 cells. A line was scratched with a plastic pipette tip through the confluent monolayer of cells maintained in serum-containing RPMI 1640 on a fibronectin-coated surface. The wounded cultures were allowed to heal for 24 h at 37 °C in the presence or absence of 25 μg/ml phaseolus vulgaris agglutinin (PHA-L) in serum-containing RPMI 1640. A: Panels show migration of cells in the presence or absence of PHAL after 24 h. B: The extent of wound closure was quantified by measurements of the width of the wound space for each case. For this value, the width was measured at twenty different locations in the wound and the mean value was compared to the width of the original closure (0 h). Values are means ± standard deviation of three separate experiments. Asterisk (*) indicates p
    Figure Legend Snippet: Effect of phaseolus vulgaris agglutinin on repair of wounds in monolayers of 92–1, Mel202, FM55P, and IGR-39 cells. A line was scratched with a plastic pipette tip through the confluent monolayer of cells maintained in serum-containing RPMI 1640 on a fibronectin-coated surface. The wounded cultures were allowed to heal for 24 h at 37 °C in the presence or absence of 25 μg/ml phaseolus vulgaris agglutinin (PHA-L) in serum-containing RPMI 1640. A: Panels show migration of cells in the presence or absence of PHAL after 24 h. B: The extent of wound closure was quantified by measurements of the width of the wound space for each case. For this value, the width was measured at twenty different locations in the wound and the mean value was compared to the width of the original closure (0 h). Values are means ± standard deviation of three separate experiments. Asterisk (*) indicates p

    Techniques Used: Transferring, Migration, Standard Deviation

    Immunodetection of α 3 , α 5 , α v , and β 1 in materials obtained after precipitation of 92–1, Mel202, FM55P, and IGR-39 cell extracts with phaseolus vulgaris agglutinin bound to agarose. One mg of the cell extracts were incubated overnight with phaseolus vulgaris agglutinin (PHA-L) immobilized on cross-linked 4% beaded agarose. Glycoproteins were released from the complexes by boiling in electrophoresis sample buffer before being subjected to 10% SDS–PAGE. Following separation, the proteins were blotted onto PVDF membrane. After being blocked the blots were incubated with one of the following antibodies specific for different integrin subunits: α 3 , α 5 , α v, and β 1 . Next, the membranes were incubated with the secondary antibodies either alkaline phosphatase conjugated goat anti-rabbit IgG (for α 3 , α 5 , α v , integrin subunits) or alkaline phosphatase coupled goat anti-mouse IgG (for β 1 integrin subunit). Visualization of immunoreactive proteins was achieved with the use of 4-nitroblue-tetrazolium salt/5-bromo-4-chloro-3-indolylophosphate solution. Lane S shows position of molecular weight markers.
    Figure Legend Snippet: Immunodetection of α 3 , α 5 , α v , and β 1 in materials obtained after precipitation of 92–1, Mel202, FM55P, and IGR-39 cell extracts with phaseolus vulgaris agglutinin bound to agarose. One mg of the cell extracts were incubated overnight with phaseolus vulgaris agglutinin (PHA-L) immobilized on cross-linked 4% beaded agarose. Glycoproteins were released from the complexes by boiling in electrophoresis sample buffer before being subjected to 10% SDS–PAGE. Following separation, the proteins were blotted onto PVDF membrane. After being blocked the blots were incubated with one of the following antibodies specific for different integrin subunits: α 3 , α 5 , α v, and β 1 . Next, the membranes were incubated with the secondary antibodies either alkaline phosphatase conjugated goat anti-rabbit IgG (for α 3 , α 5 , α v , integrin subunits) or alkaline phosphatase coupled goat anti-mouse IgG (for β 1 integrin subunit). Visualization of immunoreactive proteins was achieved with the use of 4-nitroblue-tetrazolium salt/5-bromo-4-chloro-3-indolylophosphate solution. Lane S shows position of molecular weight markers.

    Techniques Used: Immunodetection, Incubation, Electrophoresis, SDS Page, Molecular Weight

    23) Product Images from "Cell Lineage and Regional Identity of Cultured Spinal Cord Neural Stem Cells and Comparison to Brain-Derived Neural Stem Cells"

    Article Title: Cell Lineage and Regional Identity of Cultured Spinal Cord Neural Stem Cells and Comparison to Brain-Derived Neural Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004213

    Stem cell associated genes are expressed in cortical and spinal cord derived neurospheres and maintained for multiple passages: RT-PCR on E14 cortical and spinal cord derived neurospheres that had been cultured in EGF and bFGF for 2 and 8 passages. Abbreviations: Cortical (C) Spinal Cord (SC).
    Figure Legend Snippet: Stem cell associated genes are expressed in cortical and spinal cord derived neurospheres and maintained for multiple passages: RT-PCR on E14 cortical and spinal cord derived neurospheres that had been cultured in EGF and bFGF for 2 and 8 passages. Abbreviations: Cortical (C) Spinal Cord (SC).

    Techniques Used: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Cell Culture

    Regional gene expression is maintained in vitro and is cell intrinsic. RT-PCR of Emx2 and Hoxd10 expression in E14 cortical and spinal cord tissue and neurospheres at first and 13 th passage (clonal density). Emx2 and Hoxd10 expression in E14 cortical and spinal cord neurospheres that had been cultured in media that was conditioned by neurospheres from the opposing region and passaged 1 and 6 times (conditioned media).
    Figure Legend Snippet: Regional gene expression is maintained in vitro and is cell intrinsic. RT-PCR of Emx2 and Hoxd10 expression in E14 cortical and spinal cord tissue and neurospheres at first and 13 th passage (clonal density). Emx2 and Hoxd10 expression in E14 cortical and spinal cord neurospheres that had been cultured in media that was conditioned by neurospheres from the opposing region and passaged 1 and 6 times (conditioned media).

    Techniques Used: Expressing, In Vitro, Reverse Transcription Polymerase Chain Reaction, Cell Culture

    LeX negative cells from spinal cord, but not cortical derived neurospheres are neural stem cells. (A) LeX expression in a coronal section of the embryonic day 15 spinal cord; LeX (Green) Dapi (Blue); Dorsal (top) ventral (bottom). (B) Percentage of cells in secondary neurospheres derived from cortex and spinal cord that express LeX. (C) Photomicrographs of spheres generated from E14 cells following sorting for LeX expression (at second passage). LeX negative cells from cortical derived neurospheres tend to form clusters rather than round phase bright neurospheres. (D) Quantification of neurosphere formation from E14 cortical and spinal cord derived cells following sorting. Sorted cells were cultured at clonal density (1,000 cells/ml) and 10,000 cells/ml. (E) Immunocytochemistry of differentiated E14 clonal neurospheres generated by LeX expressing and non expressing cells from cortical and spinal cord derived neurospheres. Upon differentiation, clusters generated by LeX negative cells from cortical neurospheres lose cell integrity and do not generate morphologically distinct cell types. TuJ1 (Green), O4 (Blue), GFAP (Red). (F) Ability of cells within a sphere generated by LeX+ and LeX− cells to form a new neurosphere. Bars are mean±SEM of at least 3 independent experiments. * P
    Figure Legend Snippet: LeX negative cells from spinal cord, but not cortical derived neurospheres are neural stem cells. (A) LeX expression in a coronal section of the embryonic day 15 spinal cord; LeX (Green) Dapi (Blue); Dorsal (top) ventral (bottom). (B) Percentage of cells in secondary neurospheres derived from cortex and spinal cord that express LeX. (C) Photomicrographs of spheres generated from E14 cells following sorting for LeX expression (at second passage). LeX negative cells from cortical derived neurospheres tend to form clusters rather than round phase bright neurospheres. (D) Quantification of neurosphere formation from E14 cortical and spinal cord derived cells following sorting. Sorted cells were cultured at clonal density (1,000 cells/ml) and 10,000 cells/ml. (E) Immunocytochemistry of differentiated E14 clonal neurospheres generated by LeX expressing and non expressing cells from cortical and spinal cord derived neurospheres. Upon differentiation, clusters generated by LeX negative cells from cortical neurospheres lose cell integrity and do not generate morphologically distinct cell types. TuJ1 (Green), O4 (Blue), GFAP (Red). (F) Ability of cells within a sphere generated by LeX+ and LeX− cells to form a new neurosphere. Bars are mean±SEM of at least 3 independent experiments. * P

    Techniques Used: Derivative Assay, Expressing, Generated, Cell Culture, Immunocytochemistry

    Lineage relationship of spinal cord derived NSCs. LeX− NSCs derived from the spinal cord can give rise to LeX+ and LeX− NSCs. LeX− NSCs do not express markers of regional identity while LeX+ NSCs express markers indicative of spinal cord identity. LeX− cells derived from LeX+ cells are not able to generate new clonal neurospheres and are likely differentiated cells. It is not clear whether LeX− NSC must pass through a LeX expressing stage prior to differentiation.
    Figure Legend Snippet: Lineage relationship of spinal cord derived NSCs. LeX− NSCs derived from the spinal cord can give rise to LeX+ and LeX− NSCs. LeX− NSCs do not express markers of regional identity while LeX+ NSCs express markers indicative of spinal cord identity. LeX− cells derived from LeX+ cells are not able to generate new clonal neurospheres and are likely differentiated cells. It is not clear whether LeX− NSC must pass through a LeX expressing stage prior to differentiation.

    Techniques Used: Derivative Assay, Expressing

    Spinal cord derived NSCs respond to mitogens in a similar fashion to cortical derived NSCs but produce more oligodendrocytes. (A) Secondary spinal cord clonal neurosphere formation from embryonic day 11, 14, 17 and post-natal day 0, in the presence of EGF or bFGF alone and in EGF and bFGF combined. (B) The percentage of E14 clonal secondary spinal cord derived neurospheres that contain cells that express markers of neurons, astrocytes and/or oligodendrocytes; (3×) indicates neurospheres containing all 3 cell types. (C) E14 secondary clonal neurosphere formation from cortical and spinal cord derived neurospheres in differing mitogen conditions. (D) Differentiated E14 clonal secondary cortical and spinal cord derived neurospheres express markers of neurons (TuJ1- Green), oligodendrocytes (O4- Blue) and astrocytes (GFAP- red). (E) Percentage of cells expressing Tuj1 (neurons) or O4 (oligodendrocytes) present in secondary embryonic day 14 differentiated cortical and spinal cord derived neurospheres. Bars are mean±SEM of at least 3 independent experiments. * P
    Figure Legend Snippet: Spinal cord derived NSCs respond to mitogens in a similar fashion to cortical derived NSCs but produce more oligodendrocytes. (A) Secondary spinal cord clonal neurosphere formation from embryonic day 11, 14, 17 and post-natal day 0, in the presence of EGF or bFGF alone and in EGF and bFGF combined. (B) The percentage of E14 clonal secondary spinal cord derived neurospheres that contain cells that express markers of neurons, astrocytes and/or oligodendrocytes; (3×) indicates neurospheres containing all 3 cell types. (C) E14 secondary clonal neurosphere formation from cortical and spinal cord derived neurospheres in differing mitogen conditions. (D) Differentiated E14 clonal secondary cortical and spinal cord derived neurospheres express markers of neurons (TuJ1- Green), oligodendrocytes (O4- Blue) and astrocytes (GFAP- red). (E) Percentage of cells expressing Tuj1 (neurons) or O4 (oligodendrocytes) present in secondary embryonic day 14 differentiated cortical and spinal cord derived neurospheres. Bars are mean±SEM of at least 3 independent experiments. * P

    Techniques Used: Derivative Assay, Expressing

    Gene Expression in LeX+ and LeX− cells derived from E14 neurospheres: (A) Schematic of double sorting protocol. B C refer to stages at which gene expression was analyzed and D refers to clonal neurosphere forming ability of cells after two LeX sorts. (B) RT-PCR of cells immediately following first sort for LeX expression. (C) RT-PCR on neurospheres generated by LeX+ and LeX− spinal cord derived cells. (D) Clonal neurosphere forming ability of cells based on the LeX expression status at two different stages of cell culture (see panel A). First “+” or “−” refers to expression at time of 1st sort and 2nd+/−refers to second sort. * P
    Figure Legend Snippet: Gene Expression in LeX+ and LeX− cells derived from E14 neurospheres: (A) Schematic of double sorting protocol. B C refer to stages at which gene expression was analyzed and D refers to clonal neurosphere forming ability of cells after two LeX sorts. (B) RT-PCR of cells immediately following first sort for LeX expression. (C) RT-PCR on neurospheres generated by LeX+ and LeX− spinal cord derived cells. (D) Clonal neurosphere forming ability of cells based on the LeX expression status at two different stages of cell culture (see panel A). First “+” or “−” refers to expression at time of 1st sort and 2nd+/−refers to second sort. * P

    Techniques Used: Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Generated, Cell Culture

    24) Product Images from "An oncogenic role of Agrin in regulating focal adhesion integrity in hepatocellular carcinoma"

    Article Title: An oncogenic role of Agrin in regulating focal adhesion integrity in hepatocellular carcinoma

    Journal: Nature Communications

    doi: 10.1038/ncomms7184

    Agrin is frequently overexpressed in HCC. ( a ) Gene expression analysis of Agrin using microarray (Oncomine) on normal liver versus HCC patient data sets. Pre-processed expression levels are Log 2 normalized and median centred ( P values=6.22e-6 and 1.22e-7, respectively). Error bars represent s .e.m. ( b ) Western blot analysis for Agrin in a cohort of liver cancer patients in Singapore. GAPDH was used as a loading control. ( c ) Detection of circulating Agrin in the plasma samples of healthy normal individuals and HCC patients by Agrin ELISA. Data represented as mean±s.d. ( d ) A working model describing the role of Agrin in HCC. Overexpression of secreted and cell surface Agrin triggers elevated binding to its receptors Lrp4 and promotes the formation of the Agrin–Lrp4–MuSK signalling complex, which activates FAs (FAK activation), Arp2/3 associated components and cortactin generating ruffling and invadopodia. This ECM sensor activity of Agrin is critical for sustaining FAK activity, cell motility, invasiveness, matrix degradation and subsequent mesenchymal marker recruitment to cell membrane. Internalized Agrin and its complex may also mediate signalling at the endosomal compartments. Cumulatively, these Agrin-mediated events are essential for hepatic tumorigenesis.
    Figure Legend Snippet: Agrin is frequently overexpressed in HCC. ( a ) Gene expression analysis of Agrin using microarray (Oncomine) on normal liver versus HCC patient data sets. Pre-processed expression levels are Log 2 normalized and median centred ( P values=6.22e-6 and 1.22e-7, respectively). Error bars represent s .e.m. ( b ) Western blot analysis for Agrin in a cohort of liver cancer patients in Singapore. GAPDH was used as a loading control. ( c ) Detection of circulating Agrin in the plasma samples of healthy normal individuals and HCC patients by Agrin ELISA. Data represented as mean±s.d. ( d ) A working model describing the role of Agrin in HCC. Overexpression of secreted and cell surface Agrin triggers elevated binding to its receptors Lrp4 and promotes the formation of the Agrin–Lrp4–MuSK signalling complex, which activates FAs (FAK activation), Arp2/3 associated components and cortactin generating ruffling and invadopodia. This ECM sensor activity of Agrin is critical for sustaining FAK activity, cell motility, invasiveness, matrix degradation and subsequent mesenchymal marker recruitment to cell membrane. Internalized Agrin and its complex may also mediate signalling at the endosomal compartments. Cumulatively, these Agrin-mediated events are essential for hepatic tumorigenesis.

    Techniques Used: Expressing, Microarray, Western Blot, Enzyme-linked Immunosorbent Assay, Over Expression, Binding Assay, Activation Assay, Activity Assay, Marker

    Agrin regulates ruffling and invadopodia in HCC cell lines. ( a ) Representative live-cell time-lapse differential interference contrast images of control and Agrin knockdown cells at indicated time frames (in seconds) are shown. Scale bar, 10 μm. Boxed area is represented as enlarged panels. Arrows indicate membrane ruffles/protrusions. ( b ) MHCC-LM3 cells IP using Arp2/3 subunit 1B (left panel) or Agrin antibodies (middle and rightmost panels) and IgG were analysed by western blotting for Agrin, Arp2/3 subunits 1B or ArpC2p34. The blot was stripped and re-probed for Arp2/3 or Agrin. Thirty micrograms (10%) of whole-cell lysate were used as input control. Lysates from Agrin knockdown cells (right panel) were used as negative control. ( c ) Immunofluorescence analysis of control and Agrin knockdown MHCC-LM3 cells 12 h post scratch assay using goat Arp2/3 antibody and mouse Agrin antibody. Representative confocal images are shown. Panels (ii and iii) represent images of cells within the wound area and edges of wound, respectively. For panel (iv), cells were washed two times with high salt wash buffer (0.5 M NaCl) for 10 min at RT before fixation and immunostaining. Enlarged Z sections were performed at the leading edge of membrane ruffles marked by white arrows. Scale bar, 10 μm. ( d ) Cells were processed similarly as in c and immunostained with mouse anti-WASP, rabbit anti-Cdc42 and goat anti-Arp2/3. Boxed area is represented as enlarged panel inset. Scale bar, 10 μm. ( e ) Biotinylated surface proteins from control or Agrin knockdown MHCC-LM3 cells were analysed by a western blot for indicated proteins. Total cell lysate was also western blotted and β-actin served as a loading control. ( f ) Control and Agrin siRNA-transfected cells either untreated or treated with soluble Agrin (20 μg ml −1 ) for 12 h were cultured on Cy3-Gelatin for another 12 h, fixed and then stained with F-actin and DAPI. Representative confocal images are shown and invadopodia was quantified using ImageJ. Boxed areas are represented as enlarged lower panels. Arrows indicate F-actin-enriched degraded gelatin areas. Scale bar, 10 μm. At least five different fields were quantified and error bars indicate the s.d. of means of at least three independent experiments ( ***P value = 0.0005 , **P value = 0.004, respectively, Student’s t test).
    Figure Legend Snippet: Agrin regulates ruffling and invadopodia in HCC cell lines. ( a ) Representative live-cell time-lapse differential interference contrast images of control and Agrin knockdown cells at indicated time frames (in seconds) are shown. Scale bar, 10 μm. Boxed area is represented as enlarged panels. Arrows indicate membrane ruffles/protrusions. ( b ) MHCC-LM3 cells IP using Arp2/3 subunit 1B (left panel) or Agrin antibodies (middle and rightmost panels) and IgG were analysed by western blotting for Agrin, Arp2/3 subunits 1B or ArpC2p34. The blot was stripped and re-probed for Arp2/3 or Agrin. Thirty micrograms (10%) of whole-cell lysate were used as input control. Lysates from Agrin knockdown cells (right panel) were used as negative control. ( c ) Immunofluorescence analysis of control and Agrin knockdown MHCC-LM3 cells 12 h post scratch assay using goat Arp2/3 antibody and mouse Agrin antibody. Representative confocal images are shown. Panels (ii and iii) represent images of cells within the wound area and edges of wound, respectively. For panel (iv), cells were washed two times with high salt wash buffer (0.5 M NaCl) for 10 min at RT before fixation and immunostaining. Enlarged Z sections were performed at the leading edge of membrane ruffles marked by white arrows. Scale bar, 10 μm. ( d ) Cells were processed similarly as in c and immunostained with mouse anti-WASP, rabbit anti-Cdc42 and goat anti-Arp2/3. Boxed area is represented as enlarged panel inset. Scale bar, 10 μm. ( e ) Biotinylated surface proteins from control or Agrin knockdown MHCC-LM3 cells were analysed by a western blot for indicated proteins. Total cell lysate was also western blotted and β-actin served as a loading control. ( f ) Control and Agrin siRNA-transfected cells either untreated or treated with soluble Agrin (20 μg ml −1 ) for 12 h were cultured on Cy3-Gelatin for another 12 h, fixed and then stained with F-actin and DAPI. Representative confocal images are shown and invadopodia was quantified using ImageJ. Boxed areas are represented as enlarged lower panels. Arrows indicate F-actin-enriched degraded gelatin areas. Scale bar, 10 μm. At least five different fields were quantified and error bars indicate the s.d. of means of at least three independent experiments ( ***P value = 0.0005 , **P value = 0.004, respectively, Student’s t test).

    Techniques Used: Western Blot, Negative Control, Immunofluorescence, Wound Healing Assay, Immunostaining, Transfection, Cell Culture, Staining

    25) Product Images from "Study of antitumor activity in breast cell lines using silver nanoparticles produced by yeast"

    Article Title: Study of antitumor activity in breast cell lines using silver nanoparticles produced by yeast

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S75835

    Antiproliferative efficacies of biosynthesized Ag NPs produced by Cryptococcus laurentii at different concentrations. Notes: MTT assay was used on MCF7, T47D, and MCF10-A. All values are expressed as the means of the difference between optical density at 480 and 690 nm ± standard deviation. Abbreviations: Ag NPs, silver nanoparticles; OD, optical density; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
    Figure Legend Snippet: Antiproliferative efficacies of biosynthesized Ag NPs produced by Cryptococcus laurentii at different concentrations. Notes: MTT assay was used on MCF7, T47D, and MCF10-A. All values are expressed as the means of the difference between optical density at 480 and 690 nm ± standard deviation. Abbreviations: Ag NPs, silver nanoparticles; OD, optical density; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

    Techniques Used: Produced, MTT Assay, Standard Deviation

    Fluorescent microscopy images of endocytosis assay using FITC-dextran. Notes: MCF7, T47D, and MCF10-A incubated with FITC-dextran 10.000 kD (0.5 mg⋅mL −1 ) at 37°C for 0, 4, and 8 hours. Nuclear fluorescence was obtained by DAPI. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.
    Figure Legend Snippet: Fluorescent microscopy images of endocytosis assay using FITC-dextran. Notes: MCF7, T47D, and MCF10-A incubated with FITC-dextran 10.000 kD (0.5 mg⋅mL −1 ) at 37°C for 0, 4, and 8 hours. Nuclear fluorescence was obtained by DAPI. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.

    Techniques Used: Microscopy, Endocytosis Assay, Incubation, Fluorescence

    Fluorescent microscopy images to identify Ag NPs and EEA1. Notes: The colocalization between EEA1 and AgNPs was observed in the confocal analysis. The arrows show the points with colocalization. Nuclear fluorescence was obtained with DAPI. A and B are MCF7 cells; C are T47D cells. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; Ag NPs, silver nanoparticles.
    Figure Legend Snippet: Fluorescent microscopy images to identify Ag NPs and EEA1. Notes: The colocalization between EEA1 and AgNPs was observed in the confocal analysis. The arrows show the points with colocalization. Nuclear fluorescence was obtained with DAPI. A and B are MCF7 cells; C are T47D cells. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; Ag NPs, silver nanoparticles.

    Techniques Used: Microscopy, Fluorescence

    Antiproliferative efficacy of biosynthesized Ag NPs produced by Cryptococcus laurentii at different concentrations after 12 hours. Notes: MTT assay was used on dynasore treated and non-treated MCF7, T47D, and MCF10-A. All values are expressed as the means of the difference between optical density at 480 and 690 nm ± standard deviation. Abbreviations: Ag NPs, silver nanoparticles; OD, optical density; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
    Figure Legend Snippet: Antiproliferative efficacy of biosynthesized Ag NPs produced by Cryptococcus laurentii at different concentrations after 12 hours. Notes: MTT assay was used on dynasore treated and non-treated MCF7, T47D, and MCF10-A. All values are expressed as the means of the difference between optical density at 480 and 690 nm ± standard deviation. Abbreviations: Ag NPs, silver nanoparticles; OD, optical density; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

    Techniques Used: Produced, MTT Assay, Standard Deviation

    26) Product Images from "Comprehensive FISH Probe Design Tool Applied to Imaging Human Immunoglobulin Class Switch Recombination"

    Article Title: Comprehensive FISH Probe Design Tool Applied to Imaging Human Immunoglobulin Class Switch Recombination

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0051675

    Class switch recombination states detected by FISH. ( a – g ) IgM, IgG or IgE-expressing cells represented by combinations of “μ” (A–F, blue), “ ” (x y, green) or “ ” (G–K, red) FISH probes. Gray background stains ( a ) DNA in a population of pure IgM-expressing cells or ( b – g ) the expressed immunoglobulin class in cells enriched for IgG or IgE expression. Dashed lines approximate the nuclear outline. Details about the applied image processing algorithm are presented in “Materials and Methods”. ( g ) Decomposition of panel ( d ) into raw images of individual spectral channels (raw 2D data for a – f are presented in Figure S1 and raw 3D data are available in Supplementary Software, https://github.com/webfish/ ). Scale bar, 2 µm. ( h ) Each of the four classes, IgM, IgG , IgG and IgE, was studied in one to four patients (1–4). From patient 4 only IgE cells were examined. The number of scrutinized cells from each patient and the patient identifiers are inside and below the graph bars, respectively.
    Figure Legend Snippet: Class switch recombination states detected by FISH. ( a – g ) IgM, IgG or IgE-expressing cells represented by combinations of “μ” (A–F, blue), “ ” (x y, green) or “ ” (G–K, red) FISH probes. Gray background stains ( a ) DNA in a population of pure IgM-expressing cells or ( b – g ) the expressed immunoglobulin class in cells enriched for IgG or IgE expression. Dashed lines approximate the nuclear outline. Details about the applied image processing algorithm are presented in “Materials and Methods”. ( g ) Decomposition of panel ( d ) into raw images of individual spectral channels (raw 2D data for a – f are presented in Figure S1 and raw 3D data are available in Supplementary Software, https://github.com/webfish/ ). Scale bar, 2 µm. ( h ) Each of the four classes, IgM, IgG , IgG and IgE, was studied in one to four patients (1–4). From patient 4 only IgE cells were examined. The number of scrutinized cells from each patient and the patient identifiers are inside and below the graph bars, respectively.

    Techniques Used: Fluorescence In Situ Hybridization, Expressing, Software

    27) Product Images from "Non-T Cell Activation Linker (NTAL)"

    Article Title: Non-T Cell Activation Linker (NTAL)

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20021405

    Functional analysis of NTAL in LAT-defective J.CaM2.5 transfectants. (A) NTAL immunoprecipitates obtained from unstimulated (−) or anti-CD3 stimulated (+) J.CaM2.5 mutants and J.CaM2.5-NTAL transfectants were analyzed by Western blotting for the presence of the indicated molecules. The top panel corresponds to tyrosine-phosphorylated NTAL (30 kD). (B) Wild-type Jurkat, J.CaM2.5, and J.CaM2.5-NTAL transfectants were stimulated by anti-CD3 IgM mAb (added at time points indicated by arrows) and increase of cytoplasmic Ca 2+ was measured. (C) Wild-type Jurkat, J.CaM2.5, and J.CaM2.5-NTAL transfectants were stimulated by optimally diluted anti-CD3 IgM mAb and after 5 min of activation Erk1/2 was detected in the cell lysates by Western blotting using anti-phospho Erk antibody; bottom panel represents control staining by anti-Erk. (D) J.CaM2.5 cells transiently transfected with the indicated FLAG-tagged constructs were stimulated for 2 min by anti-CD3 and anti-CD28 mAbs and activation of Erk1/2 was detected as in C (top panel); presence of equal amounts of Erk1/2 in all samples was ascertained (middle panel) and the level of expression of individual FLAG-tagged proteins was determined (bottom panel).
    Figure Legend Snippet: Functional analysis of NTAL in LAT-defective J.CaM2.5 transfectants. (A) NTAL immunoprecipitates obtained from unstimulated (−) or anti-CD3 stimulated (+) J.CaM2.5 mutants and J.CaM2.5-NTAL transfectants were analyzed by Western blotting for the presence of the indicated molecules. The top panel corresponds to tyrosine-phosphorylated NTAL (30 kD). (B) Wild-type Jurkat, J.CaM2.5, and J.CaM2.5-NTAL transfectants were stimulated by anti-CD3 IgM mAb (added at time points indicated by arrows) and increase of cytoplasmic Ca 2+ was measured. (C) Wild-type Jurkat, J.CaM2.5, and J.CaM2.5-NTAL transfectants were stimulated by optimally diluted anti-CD3 IgM mAb and after 5 min of activation Erk1/2 was detected in the cell lysates by Western blotting using anti-phospho Erk antibody; bottom panel represents control staining by anti-Erk. (D) J.CaM2.5 cells transiently transfected with the indicated FLAG-tagged constructs were stimulated for 2 min by anti-CD3 and anti-CD28 mAbs and activation of Erk1/2 was detected as in C (top panel); presence of equal amounts of Erk1/2 in all samples was ascertained (middle panel) and the level of expression of individual FLAG-tagged proteins was determined (bottom panel).

    Techniques Used: Functional Assay, Western Blot, Activation Assay, Staining, Transfection, Construct, Expressing

    Tissue and subcellular localization of NTAL. (A) Paraffin section of lymphoid tissue immunoperoxidase stained for NTAL; the major positive structures are germinal centers. (B) Localization of NTAL in buoyant detergent-resistant microdomains (GEMs). THP-1 cells were solubilized in the presence of 3% nonionic detergent Brij-58 or 1% laurylmaltoside (LM; a detergent known to disrupt GEMs) and subjected to sucrose density gradient ultracentrifugation; the fractions (numbered from top to bottom) were analyzed by Western blotting. (C) Biosynthetic labeling of NTAL with [ 3 H]palmitate; NTAL immunoprecipitate was analyzed by SDS-PAGE followed by fluorography of the gel. (D) Plasma membrane localization of NTAL (green) as determined by confocal microscopy in THP-1 cells and J.CaM2.5-NTAL transfectants; nuclei are shown in red.
    Figure Legend Snippet: Tissue and subcellular localization of NTAL. (A) Paraffin section of lymphoid tissue immunoperoxidase stained for NTAL; the major positive structures are germinal centers. (B) Localization of NTAL in buoyant detergent-resistant microdomains (GEMs). THP-1 cells were solubilized in the presence of 3% nonionic detergent Brij-58 or 1% laurylmaltoside (LM; a detergent known to disrupt GEMs) and subjected to sucrose density gradient ultracentrifugation; the fractions (numbered from top to bottom) were analyzed by Western blotting. (C) Biosynthetic labeling of NTAL with [ 3 H]palmitate; NTAL immunoprecipitate was analyzed by SDS-PAGE followed by fluorography of the gel. (D) Plasma membrane localization of NTAL (green) as determined by confocal microscopy in THP-1 cells and J.CaM2.5-NTAL transfectants; nuclei are shown in red.

    Techniques Used: Paraffin Section, Staining, Western Blot, Labeling, SDS Page, Confocal Microscopy

    Expression of NTAL. (A) cDNA encoding human NTAL was expressed in J.CaM2.5 cells and the protein product was visualized by Western blotting of the transfectants detergent lysate as compared with Ramos cells (expressing endogenous NTAL). (B) Western blotting of the indicated subpopulations of human peripheral blood cells (immunostaining for NTAL or Erk; the latter was used as a loading control).
    Figure Legend Snippet: Expression of NTAL. (A) cDNA encoding human NTAL was expressed in J.CaM2.5 cells and the protein product was visualized by Western blotting of the transfectants detergent lysate as compared with Ramos cells (expressing endogenous NTAL). (B) Western blotting of the indicated subpopulations of human peripheral blood cells (immunostaining for NTAL or Erk; the latter was used as a loading control).

    Techniques Used: Expressing, Western Blot, Immunostaining

    28) Product Images from "Divergent roles for Eph and Ephrin in Avian Cranial Neural Crest"

    Article Title: Divergent roles for Eph and Ephrin in Avian Cranial Neural Crest

    Journal: BMC Developmental Biology

    doi: 10.1186/1471-213X-8-56

    Binding of ephrin-B1 and EphB2 affinity probes to cultured avian CNC cells. (a) Cells exposed to Fc protein are not labeled by an anti-Fc antibody. (b) Bright peripheral spots of anti-Fc staining result from exposure of cells to ephrin-B1/Fc protein (arrowheads). (c) Cells exposed to EphB2/Fc protein exhibit weaker anti-Fc staining at points of contact between neighboring cells (arrowheads). Insets show HNK-1 staining for each field of cells.
    Figure Legend Snippet: Binding of ephrin-B1 and EphB2 affinity probes to cultured avian CNC cells. (a) Cells exposed to Fc protein are not labeled by an anti-Fc antibody. (b) Bright peripheral spots of anti-Fc staining result from exposure of cells to ephrin-B1/Fc protein (arrowheads). (c) Cells exposed to EphB2/Fc protein exhibit weaker anti-Fc staining at points of contact between neighboring cells (arrowheads). Insets show HNK-1 staining for each field of cells.

    Techniques Used: Binding Assay, Cell Culture, Labeling, Staining

    Outgrowth of cranial neural crest cells from stage 10–12 chicken neural tube explants onto ephrin-B1 or EphB2 stripe assay substrates in the presence of soluble competitors. Representative results from the four experimental conditions. The position of the first set of lanes is revealed by an FITC marker (faint green stripes). Cells have been double labeled with HNK-1 antibody (red) and DAPI nuclear stain (blue). All experiments were done with a stripe protein concentration of 64 μg/ml. (a). Clustered ephrin-B1/Fc + FN vs. FN with soluble Fc. Cell outgrowth from the neural tube is strongly localized to the lanes between stripes of FITC-marked ephrin-B1/Fc. (b) Clustered ephrin-B1/Fc + FN vs. FN with soluble ephrin-B1/Fc. Substitution of the soluble Fc with ephrin-B1/Fc results in an increase in the number of cells found on the stripes of immobilized ephrin-B1/Fc. (c) Clustered EphB2/Fc + FN vs. FN with soluble Fc and (d) clustered EphB2/Fc + FN vs. FN with soluble EphB2/Fc. As with the ephrin-B1/Fc experiments, there is a significant increase in the number of cells on lanes of substrate-bound EphB2/Fc in going from adding soluble Fc to soluble EphB2/Fc to the culture medium.
    Figure Legend Snippet: Outgrowth of cranial neural crest cells from stage 10–12 chicken neural tube explants onto ephrin-B1 or EphB2 stripe assay substrates in the presence of soluble competitors. Representative results from the four experimental conditions. The position of the first set of lanes is revealed by an FITC marker (faint green stripes). Cells have been double labeled with HNK-1 antibody (red) and DAPI nuclear stain (blue). All experiments were done with a stripe protein concentration of 64 μg/ml. (a). Clustered ephrin-B1/Fc + FN vs. FN with soluble Fc. Cell outgrowth from the neural tube is strongly localized to the lanes between stripes of FITC-marked ephrin-B1/Fc. (b) Clustered ephrin-B1/Fc + FN vs. FN with soluble ephrin-B1/Fc. Substitution of the soluble Fc with ephrin-B1/Fc results in an increase in the number of cells found on the stripes of immobilized ephrin-B1/Fc. (c) Clustered EphB2/Fc + FN vs. FN with soluble Fc and (d) clustered EphB2/Fc + FN vs. FN with soluble EphB2/Fc. As with the ephrin-B1/Fc experiments, there is a significant increase in the number of cells on lanes of substrate-bound EphB2/Fc in going from adding soluble Fc to soluble EphB2/Fc to the culture medium.

    Techniques Used: Stripping Membranes, Marker, Labeling, Staining, Protein Concentration

    Distribution of EphB2 mRNA and HNK-1 protein in the embryonic avian hindbrain. Wholemount (a-c) and cross-section (d-f) images of a stage 14 chicken embryo double labeled for EphB2 and HNK-1. (a-c) EphB2 probe staining is in patches (arrows) bordering cells labeled by HNK-1. Inset shows embryo at lower magnification. The margins of the regions expressing EphB2 have been traced with a thin white line in panel a. The tracing from panel a has been superimposed on the image in panel b to show the margins relative to the HNK-1 expressing cells. (d-fi) In cross-section (taken at black line in c), probe (arrow) and HNK-1 stained cells have a non-overlapping distribution. Inset shows section at lower magnification. Ec, ectoderm; My, mesenchyme; s, somite.
    Figure Legend Snippet: Distribution of EphB2 mRNA and HNK-1 protein in the embryonic avian hindbrain. Wholemount (a-c) and cross-section (d-f) images of a stage 14 chicken embryo double labeled for EphB2 and HNK-1. (a-c) EphB2 probe staining is in patches (arrows) bordering cells labeled by HNK-1. Inset shows embryo at lower magnification. The margins of the regions expressing EphB2 have been traced with a thin white line in panel a. The tracing from panel a has been superimposed on the image in panel b to show the margins relative to the HNK-1 expressing cells. (d-fi) In cross-section (taken at black line in c), probe (arrow) and HNK-1 stained cells have a non-overlapping distribution. Inset shows section at lower magnification. Ec, ectoderm; My, mesenchyme; s, somite.

    Techniques Used: Labeling, Staining, Expressing

    Outgrowth of cranial neural crest cells from stage 10–12 chicken neural tube explants onto ephrin-B1 or EphB2 stripe assay substrates. Representative results from the three experimental conditions. The position of the first set of lanes is revealed by an FITC marker (faint green stripes). In the last image on each row (c, f, j), cells have been double labeled with HNK-1 antibody (red) and DAPI nuclear stain (blue). (a-c) Clustered Fc + FN vs. FN. Cell outgrowth shows no bias for either set of lanes, regardless of Fc protein concentration. (d – f) Clustered ephrin-B1/Fc + FN vs. FN and (g – j) clustered EphB2/Fc + FN vs. FN. Cell outgrowth is even at low stripe protein concentrations, but becomes more restricted to the lanes between the FITC marked stripes with increasing concentrations in the coating solution.
    Figure Legend Snippet: Outgrowth of cranial neural crest cells from stage 10–12 chicken neural tube explants onto ephrin-B1 or EphB2 stripe assay substrates. Representative results from the three experimental conditions. The position of the first set of lanes is revealed by an FITC marker (faint green stripes). In the last image on each row (c, f, j), cells have been double labeled with HNK-1 antibody (red) and DAPI nuclear stain (blue). (a-c) Clustered Fc + FN vs. FN. Cell outgrowth shows no bias for either set of lanes, regardless of Fc protein concentration. (d – f) Clustered ephrin-B1/Fc + FN vs. FN and (g – j) clustered EphB2/Fc + FN vs. FN. Cell outgrowth is even at low stripe protein concentrations, but becomes more restricted to the lanes between the FITC marked stripes with increasing concentrations in the coating solution.

    Techniques Used: Stripping Membranes, Marker, Labeling, Staining, Protein Concentration

    Bar graph summarizing the stripe assay results for the outgrowth of cranial neural crest cells from stage 10–12 chicken neural tube explants onto ephrin-B1 or EphB2 in the presence of soluble competitors. There are significantly more cells found growing on lanes of substrate bound ephrin-B1/Fc or EphB2/Fc protein in the presence of soluble competitor than in the presence of soluble Fc.
    Figure Legend Snippet: Bar graph summarizing the stripe assay results for the outgrowth of cranial neural crest cells from stage 10–12 chicken neural tube explants onto ephrin-B1 or EphB2 in the presence of soluble competitors. There are significantly more cells found growing on lanes of substrate bound ephrin-B1/Fc or EphB2/Fc protein in the presence of soluble competitor than in the presence of soluble Fc.

    Techniques Used: Stripping Membranes

    Bar graph summarizing the quantified stripe assay results for the outgrowth of cranial neural crest cells from stage 10–12 chicken neural tube explants onto ephrin-B1 or EphB2 stripe assay substrates (64 μg/ml coating concentration).
    Figure Legend Snippet: Bar graph summarizing the quantified stripe assay results for the outgrowth of cranial neural crest cells from stage 10–12 chicken neural tube explants onto ephrin-B1 or EphB2 stripe assay substrates (64 μg/ml coating concentration).

    Techniques Used: Stripping Membranes, Concentration Assay

    29) Product Images from "Functional neural differentiation of human adipose tissue-derived stem cells using bFGF and forskolin"

    Article Title: Functional neural differentiation of human adipose tissue-derived stem cells using bFGF and forskolin

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-11-25

    Analysis of cell type-specific markers in NI-hADSCs . Human ADSCs were induced to differentiate into neural cells in the presence of bFGF and forskolin over two weeks. a , Fluorescent immunocytochemistry revealed that the expression of nestin, Tuj1, MAP2, NFL, NFM, NFH, NSE, NeuN, GAP43, SNAP25, GFAP, and CNPase in NI-hADSCs increased more than those of primary hADSCs. Scale bars measure 50 μm. b , Immunocytochemical data depicts the high ratio of NI-hADSCs expression above neuronal markers. The number of positive cells was counted and the ratio of positive cells to nuclei was analyzed for each antigen (n = 7). c and d , RT-PCR analysis demonstrated increased mRNA expression for ABCG2, nestin, Tuj1, MAP2, NFL, NFM, NSE, GAP43, SNAP25, GFAP and CNPase genes. GAPDH was used as a control. The intensity of each gene was normalized to GAPDH and these results were repeated at least five times. * P
    Figure Legend Snippet: Analysis of cell type-specific markers in NI-hADSCs . Human ADSCs were induced to differentiate into neural cells in the presence of bFGF and forskolin over two weeks. a , Fluorescent immunocytochemistry revealed that the expression of nestin, Tuj1, MAP2, NFL, NFM, NFH, NSE, NeuN, GAP43, SNAP25, GFAP, and CNPase in NI-hADSCs increased more than those of primary hADSCs. Scale bars measure 50 μm. b , Immunocytochemical data depicts the high ratio of NI-hADSCs expression above neuronal markers. The number of positive cells was counted and the ratio of positive cells to nuclei was analyzed for each antigen (n = 7). c and d , RT-PCR analysis demonstrated increased mRNA expression for ABCG2, nestin, Tuj1, MAP2, NFL, NFM, NSE, GAP43, SNAP25, GFAP and CNPase genes. GAPDH was used as a control. The intensity of each gene was normalized to GAPDH and these results were repeated at least five times. * P

    Techniques Used: Immunocytochemistry, Expressing, Reverse Transcription Polymerase Chain Reaction

    30) Product Images from "Lipid Oxidation Induced by RF Waves and Mediated by Ferritin Iron Causes Activation of Ferritin-Tagged Ion Channels"

    Article Title: Lipid Oxidation Induced by RF Waves and Mediated by Ferritin Iron Causes Activation of Ferritin-Tagged Ion Channels

    Journal: Cell reports

    doi: 10.1016/j.celrep.2020.02.070

    Iron, ROS, Lipids, and Oxidized Lipids Are Involved in RF-Induced Activation of TRPV FeRIC (A and D) Average changes (± SEM) in GCaMP6 ΔF/F0 in (A) N2a cells expressing TRPV4 FeRIC or (D) HEK293T cells expressing TRPV1 FeRIC following exposure to RF (12 μT, gray rectangle) and next chemical agonists (bar). In separate series, cells were incubated for 15–60 min before RF and agonist stimulation with PIH, TROLOX, ACA, NAC, or DPPD. (B and E) Averages of the GCaMP6 AUC (± SEM) for the period of RF stimulation. (C and F) Cell responsiveness (± SEM) for data in (B) and (E), respectively. (G and H) Average changes (± SEM) in GCaMP6 ΔF/F0 in N2a cells expressing GCaMP6 or expressing GCaMP6 plus TRPV4 FeRIC or TRPV4 WT following exposure to (G) Fe 2+ (100 μM) or (H) H 2 O 2 (100 μM) (black arrows) and next GSK101 (bar). Insets: GCaMP6 AUC (± SEM) for the period from 61 to 290 s. In separate series, cells were incubated for 15 min before stimulation with GSK219. (I) Normalized abundances of lipids detected in N2a cells expressing TRPV4 FeRIC or TRPV4 WT that were stimulated with RF, as measured by LC-MS. Ion abundances were normalized to the average ion abundance for samples that were not stimulated with RF. Data indicate means (± SEM) from 3–4 separate experiments each for RF and no RF conditions. (J and M) Average changes (± SEM) in GCaMP6 ΔF/F0 in cells expressing (J) TRPV4 FeRIC or (M) TRPV1 FeRIC following exposure to AA, 5,6-EEA, 4-HNE, NEM, and OxPAPC (black arrows) and next agonists (bar). (K and N) Averages of the GCaMP6 AUC (± SEM) for the period from 61 to 290 s. (L and O) Cell responsiveness (± SEM) for data in (K) and (N), respectively. GCaMP6 data correspond to 3–7 independent experiments with 62–276 cells analyzed. For LC-MS data, the statistical test applied was Student”s t test. Where applicable, *p
    Figure Legend Snippet: Iron, ROS, Lipids, and Oxidized Lipids Are Involved in RF-Induced Activation of TRPV FeRIC (A and D) Average changes (± SEM) in GCaMP6 ΔF/F0 in (A) N2a cells expressing TRPV4 FeRIC or (D) HEK293T cells expressing TRPV1 FeRIC following exposure to RF (12 μT, gray rectangle) and next chemical agonists (bar). In separate series, cells were incubated for 15–60 min before RF and agonist stimulation with PIH, TROLOX, ACA, NAC, or DPPD. (B and E) Averages of the GCaMP6 AUC (± SEM) for the period of RF stimulation. (C and F) Cell responsiveness (± SEM) for data in (B) and (E), respectively. (G and H) Average changes (± SEM) in GCaMP6 ΔF/F0 in N2a cells expressing GCaMP6 or expressing GCaMP6 plus TRPV4 FeRIC or TRPV4 WT following exposure to (G) Fe 2+ (100 μM) or (H) H 2 O 2 (100 μM) (black arrows) and next GSK101 (bar). Insets: GCaMP6 AUC (± SEM) for the period from 61 to 290 s. In separate series, cells were incubated for 15 min before stimulation with GSK219. (I) Normalized abundances of lipids detected in N2a cells expressing TRPV4 FeRIC or TRPV4 WT that were stimulated with RF, as measured by LC-MS. Ion abundances were normalized to the average ion abundance for samples that were not stimulated with RF. Data indicate means (± SEM) from 3–4 separate experiments each for RF and no RF conditions. (J and M) Average changes (± SEM) in GCaMP6 ΔF/F0 in cells expressing (J) TRPV4 FeRIC or (M) TRPV1 FeRIC following exposure to AA, 5,6-EEA, 4-HNE, NEM, and OxPAPC (black arrows) and next agonists (bar). (K and N) Averages of the GCaMP6 AUC (± SEM) for the period from 61 to 290 s. (L and O) Cell responsiveness (± SEM) for data in (K) and (N), respectively. GCaMP6 data correspond to 3–7 independent experiments with 62–276 cells analyzed. For LC-MS data, the statistical test applied was Student”s t test. Where applicable, *p

    Techniques Used: Activation Assay, Expressing, Incubation, Liquid Chromatography with Mass Spectroscopy

    31) Product Images from "Modular and distinct PlexinA4/Farp2/Rac1 signaling controls dendrite morphogenesis"

    Article Title: Modular and distinct PlexinA4/Farp2/Rac1 signaling controls dendrite morphogenesis

    Journal: bioRxiv

    doi: 10.1101/2020.01.15.908434

    PlxnA4 KRK-AAA and Farp2 KO DRG axons show intact Sema3A-dependent responses in vitro . A-H , DRG explants from WT ( A-B ), PlxnA4 KRK-AAA ( C-D ) and Farp2 KO ( F-G ) E13.5 embryos were grown for 48hr, treated with 0.1, 0.5 or 1nM AP-Sema3A (only 1nM is shown) or control conditioned media and stained using Phalloidin-Rhodamine to visualize growth cone collapse. Arrows indicate intact growth cones and arrowheads indicate collapsed growth cones. Quantification of collapse response ( E and H ) as a mean percentage of collapsed growth cones out of the total ± SEM; N.S., non-significant; two-way ANOVA with post hoc Tukey test. Scale bar, 50μm. I-M , Schematic representation of the collagen axonal repulsion assay ( I ), where E13.5 DRG explants from PlxnA4 KRK-AAA and WT littermates ( J-K ) or Farp2 KO and WT littermates ( L-M ) were co-cultured for 48hrs with a COS1 aggregate either secreting myc-Sema3A (dashed circle) or expressing control PAY1-GFP (green). Cultures were visualized using anti-Tubulin class III immuno-staining. N , Quantification of axonal repulsion using the proximal/distal (P/D) ratio, as indicated in ( I ). Data are means ± SEM; N.S., non-significant, Student’s t-test. Scale bar, 500μm.
    Figure Legend Snippet: PlxnA4 KRK-AAA and Farp2 KO DRG axons show intact Sema3A-dependent responses in vitro . A-H , DRG explants from WT ( A-B ), PlxnA4 KRK-AAA ( C-D ) and Farp2 KO ( F-G ) E13.5 embryos were grown for 48hr, treated with 0.1, 0.5 or 1nM AP-Sema3A (only 1nM is shown) or control conditioned media and stained using Phalloidin-Rhodamine to visualize growth cone collapse. Arrows indicate intact growth cones and arrowheads indicate collapsed growth cones. Quantification of collapse response ( E and H ) as a mean percentage of collapsed growth cones out of the total ± SEM; N.S., non-significant; two-way ANOVA with post hoc Tukey test. Scale bar, 50μm. I-M , Schematic representation of the collagen axonal repulsion assay ( I ), where E13.5 DRG explants from PlxnA4 KRK-AAA and WT littermates ( J-K ) or Farp2 KO and WT littermates ( L-M ) were co-cultured for 48hrs with a COS1 aggregate either secreting myc-Sema3A (dashed circle) or expressing control PAY1-GFP (green). Cultures were visualized using anti-Tubulin class III immuno-staining. N , Quantification of axonal repulsion using the proximal/distal (P/D) ratio, as indicated in ( I ). Data are means ± SEM; N.S., non-significant, Student’s t-test. Scale bar, 500μm.

    Techniques Used: In Vitro, Staining, Cell Culture, Expressing, Immunostaining

    Inhibition of Rac signaling abolishes Sema3A-mediated cortical neuron dendrite elaboration but does not hinder Sema3A-dependent growth cone collapse of WT or PlxnA4 KRK-AAA DRG axons in vitro . A-L , DRG explants from WT ( A-F ) and PlxnA4 KRK-AAA littermates ( G-L ) E13.5 embryos were grown for 48hr, treated for 30min with the pan-Rac inhibitor EHT 1864 at a concentration of 5μM ( B, D, H, J ), 10μM ( C, E, I, K ) or nothing as control ( A, G, F, L ). Then, 1nM Sema3A or control conditioned media was added for 30min ( D-F, J-L and A-C, G-I , respectively), followed by fixation and Phalloidin-Rhodamine staining for assessment of growth cone collapse. Black arrows indicate intact growth cones and arrowheads indicate collapsed growth cones. M-N , Quantification of collapse response as a mean percentage of collapsed growth cones out of the total ± SEM in WT and PlxnA4 KRK-AAA axons, respectively. N.S. – non-significant. Scale bar, 50μm. O , Representative confocal micrographs of dissociated primary cortical neurons obtained from WT E13.5 embryos. The neurons were treated with 5nM AP, treated with 5nM Sema3A, 5nM Sema3A+2.5uM EHT, 5nM Sema3A+5uM EHT, and 5nM Sema3A+10uM EHT. P-S , Sholl analysis of dendritic intersections ( P ) total dendritic length ( Q ) the DCI ( R ) and number of dendritic tips ( S ), for all treatment conditions described above. Data are means, ±SEM from n=3 independent cultures, two-way ANOVA with post doc Tukey test, p
    Figure Legend Snippet: Inhibition of Rac signaling abolishes Sema3A-mediated cortical neuron dendrite elaboration but does not hinder Sema3A-dependent growth cone collapse of WT or PlxnA4 KRK-AAA DRG axons in vitro . A-L , DRG explants from WT ( A-F ) and PlxnA4 KRK-AAA littermates ( G-L ) E13.5 embryos were grown for 48hr, treated for 30min with the pan-Rac inhibitor EHT 1864 at a concentration of 5μM ( B, D, H, J ), 10μM ( C, E, I, K ) or nothing as control ( A, G, F, L ). Then, 1nM Sema3A or control conditioned media was added for 30min ( D-F, J-L and A-C, G-I , respectively), followed by fixation and Phalloidin-Rhodamine staining for assessment of growth cone collapse. Black arrows indicate intact growth cones and arrowheads indicate collapsed growth cones. M-N , Quantification of collapse response as a mean percentage of collapsed growth cones out of the total ± SEM in WT and PlxnA4 KRK-AAA axons, respectively. N.S. – non-significant. Scale bar, 50μm. O , Representative confocal micrographs of dissociated primary cortical neurons obtained from WT E13.5 embryos. The neurons were treated with 5nM AP, treated with 5nM Sema3A, 5nM Sema3A+2.5uM EHT, 5nM Sema3A+5uM EHT, and 5nM Sema3A+10uM EHT. P-S , Sholl analysis of dendritic intersections ( P ) total dendritic length ( Q ) the DCI ( R ) and number of dendritic tips ( S ), for all treatment conditions described above. Data are means, ±SEM from n=3 independent cultures, two-way ANOVA with post doc Tukey test, p

    Techniques Used: Inhibition, In Vitro, Concentration Assay, Staining

    Differential requirement for the KRK motif and its downstream signaling effector Farp2 in Plexin-A4-mediated cellular responses. A , Sema3A-Neuropilin1/PlexinA4 signaling promotes basal dendrite elaboration in deep-layer pyramidal cortical neurons on one hand and growth cone collapse and axonal repulsion on the other. B , Substitution of the KRK motif of Plexin-A4 to AAA, ablation of the Plexin-A4-binding effector, the Rac1 GEF Farp2, or inhibition of Rac1 specifically abrogate dendrite elaboration but not growth cone collapse and axonal repulsion.
    Figure Legend Snippet: Differential requirement for the KRK motif and its downstream signaling effector Farp2 in Plexin-A4-mediated cellular responses. A , Sema3A-Neuropilin1/PlexinA4 signaling promotes basal dendrite elaboration in deep-layer pyramidal cortical neurons on one hand and growth cone collapse and axonal repulsion on the other. B , Substitution of the KRK motif of Plexin-A4 to AAA, ablation of the Plexin-A4-binding effector, the Rac1 GEF Farp2, or inhibition of Rac1 specifically abrogate dendrite elaboration but not growth cone collapse and axonal repulsion.

    Techniques Used: Binding Assay, Inhibition

    32) Product Images from "Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays"

    Article Title: Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.051219

    Titers of Spike-pseudotyped lentiviral particles in 293T-ACE2 cells. ( A ) Titers of the ZsGreen backbone pseudotyped with the three Spike variants or VSV G, as determined by counting green cells via flow cytometry analysis at 48 hours post-infection, and then calculating transduction-competent viral particles per ml from the percentage of green cells. The “n.d.” indicates that the titer was not detectable. ( B ) Titers of the Luciferase-IRES-ZsGreen backbone as determined by measuring relative luciferase units (RLUs). RLUs were determined at 48 hours after infecting ∼2.3×10 4 293T-ACE2 cells per well in 96-well plates. The RLUs per mL for the Spike-pseudotyped viruses are the average of three 3-fold serial dilutions of virus starting at 50 µL virus in a total volume of 150 µL. For the VSV G-pseudotyped virus, RLUs per mL were averaged from two 3-fold dilutions starting at 3 µL virus in a total volume of 150 µL. ( C ) Microscope images showing 293T-ACE2 cells transduced with Spike pseudotyped virus with either the ZsGreen or Luciferase-IRES-ZsGreen backbone at 60 hours post-infection. As can be seen from the images, the ZsGreen backbone gives a stronger fluorescent signal than the Luciferase-IRES-ZsGreen backbone, presumably because this protein is expressed more strongly as the sole CMV-promoter driven transcript than as the second transcript driven by an IRES.
    Figure Legend Snippet: Titers of Spike-pseudotyped lentiviral particles in 293T-ACE2 cells. ( A ) Titers of the ZsGreen backbone pseudotyped with the three Spike variants or VSV G, as determined by counting green cells via flow cytometry analysis at 48 hours post-infection, and then calculating transduction-competent viral particles per ml from the percentage of green cells. The “n.d.” indicates that the titer was not detectable. ( B ) Titers of the Luciferase-IRES-ZsGreen backbone as determined by measuring relative luciferase units (RLUs). RLUs were determined at 48 hours after infecting ∼2.3×10 4 293T-ACE2 cells per well in 96-well plates. The RLUs per mL for the Spike-pseudotyped viruses are the average of three 3-fold serial dilutions of virus starting at 50 µL virus in a total volume of 150 µL. For the VSV G-pseudotyped virus, RLUs per mL were averaged from two 3-fold dilutions starting at 3 µL virus in a total volume of 150 µL. ( C ) Microscope images showing 293T-ACE2 cells transduced with Spike pseudotyped virus with either the ZsGreen or Luciferase-IRES-ZsGreen backbone at 60 hours post-infection. As can be seen from the images, the ZsGreen backbone gives a stronger fluorescent signal than the Luciferase-IRES-ZsGreen backbone, presumably because this protein is expressed more strongly as the sole CMV-promoter driven transcript than as the second transcript driven by an IRES.

    Techniques Used: Flow Cytometry, Infection, Transduction, Luciferase, Microscopy

    33) Product Images from "Precise, high-throughput production of multicellular spheroids with a bespoke 3D bioprinter"

    Article Title: Precise, high-throughput production of multicellular spheroids with a bespoke 3D bioprinter

    Journal: bioRxiv

    doi: 10.1101/2020.04.06.028548

    3D bioprinted spheroids have a similar organization as manually produced spheroids. ( A ) 3D rendered and 3D cross-sectioned ( via optical sectioning) light-sheet microscopy images of the 3D bioprinted (left) and manual SK-N-BE( 2 ) spheroids, labelled with α-Ki67 antibody (green), indicating cell proliferation and the DNA dye Hoechst 33342 (blue). Scale bars = 200 µm. ( B ) Percentage of HIF1α positive, ( C ) cleaved caspase-3 positive and ( D) CD133 positive cells as determined by FACS. (In B-D , n = 3, Unpaired t-test; n.s.) ( E ) Lattice light-sheet images of a bioprinted spheroid, stained with phalloidin-568 (red) and SYTOX green (blue). The images were further sliced at 34.95 µm ( E1 ), 27.45 µm ( E2 ) and 19.95 µm ( E3 ) from the top to show the high-resolution cellular arrangement of the spheroids. Scale bars = 10 µm. ( F ) Quantification of the cell-cell density was conducted by measuring the average distance between the nearest neighbouring nuclei in 3-dimension for both manually prepared and 3D bioprinted SK-N-BE( 2 ) spheroids ( n = 3). Results are means ± SEM
    Figure Legend Snippet: 3D bioprinted spheroids have a similar organization as manually produced spheroids. ( A ) 3D rendered and 3D cross-sectioned ( via optical sectioning) light-sheet microscopy images of the 3D bioprinted (left) and manual SK-N-BE( 2 ) spheroids, labelled with α-Ki67 antibody (green), indicating cell proliferation and the DNA dye Hoechst 33342 (blue). Scale bars = 200 µm. ( B ) Percentage of HIF1α positive, ( C ) cleaved caspase-3 positive and ( D) CD133 positive cells as determined by FACS. (In B-D , n = 3, Unpaired t-test; n.s.) ( E ) Lattice light-sheet images of a bioprinted spheroid, stained with phalloidin-568 (red) and SYTOX green (blue). The images were further sliced at 34.95 µm ( E1 ), 27.45 µm ( E2 ) and 19.95 µm ( E3 ) from the top to show the high-resolution cellular arrangement of the spheroids. Scale bars = 10 µm. ( F ) Quantification of the cell-cell density was conducted by measuring the average distance between the nearest neighbouring nuclei in 3-dimension for both manually prepared and 3D bioprinted SK-N-BE( 2 ) spheroids ( n = 3). Results are means ± SEM

    Techniques Used: Produced, Microscopy, FACS, Staining

    34) Product Images from "Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells"

    Article Title: Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells

    Journal: bioRxiv

    doi: 10.1101/2020.02.25.963017

    Hyperpolarization improves CPP uptake  in vivo . (A)  Assessment of CPP  in vivo  uptake in zebrafish embryos in normal and hyperpolarized conditions. Forty-eight hour post fertilization, zebrafish embryos were injected with 3.12 mM FITC-TAT-RasGAP 317-326 (W317A) with or without 10 mM valinomycin. Scale bar: 200 mm. Comparison between conditions in the presence or in the absence of valinomycin was done using two-tailed paired t-test. The results correspond to three independent experiments.  (B)  Assessment of CPP  in vivo  uptake in C57BL/6N mice in normal and hyperpolarized conditions. Mice were injected with 5 mM FITC-TAT-RasGAP 317-326 (W317A) with or without 10 mM valinomycin (n=11 injections per condition). Comparison between conditions in the presence or in the absence of valinomycin was done using two-tailed paired t-test.
    Figure Legend Snippet: Hyperpolarization improves CPP uptake in vivo . (A) Assessment of CPP in vivo uptake in zebrafish embryos in normal and hyperpolarized conditions. Forty-eight hour post fertilization, zebrafish embryos were injected with 3.12 mM FITC-TAT-RasGAP 317-326 (W317A) with or without 10 mM valinomycin. Scale bar: 200 mm. Comparison between conditions in the presence or in the absence of valinomycin was done using two-tailed paired t-test. The results correspond to three independent experiments. (B) Assessment of CPP in vivo uptake in C57BL/6N mice in normal and hyperpolarized conditions. Mice were injected with 5 mM FITC-TAT-RasGAP 317-326 (W317A) with or without 10 mM valinomycin (n=11 injections per condition). Comparison between conditions in the presence or in the absence of valinomycin was done using two-tailed paired t-test.

    Techniques Used: In Vivo, Injection, Two Tailed Test, Mouse Assay

    Related to Fig. 5. Zebrafish and mouse membrane potential modulation. (A)  Eighteen hours post fertilization zebrafish embryos were incubated 40 minutes with the indicated concentrations of valinomycin with 950 nM DiBac4(3). DiBac4(3) fluorescence was then recorded and normalized to the non-treated control. The decrease in DiBac4(3) fluorescence indicates membrane hyperpolarization. Membrane potential values could not be calculated, as a standard curve would have to be performed in zebrafish. DiBac4(3) uptake was assessed from confocal images of the fish tail region.  (B)  Eighteen hours post fertilization zebrafish embryos were incubated with or without 3.12 mM TAT-RasGAP 317-326  (W317A), a mutant version that is not toxic to cells, in the presence of the indicated concentrations of valinomycin. Peptide uptake was assessed from confocal images of the fish tail region.  (C-D)  Eighteen hours post fertilization zebrafish embryos were incubated 1 hour with the indicated concentrations of valinomycin, in the absence (panel C) or in the presence (panel D) of 3.12 mM TAT-RasGAP 317-326  (W317A), then washed, and incubated in Egg water. The viability of each fish was assessed over 52 hours at the indicated time points.  (E)  Representative images of zebrafish treated as described in panel C and D, washed, and further incubated in Egg water. Images were taken with a CYTATION3 apparatus at a 4x magnification.  (F)  Survival of 48 hours post fertilization zebrafish embryos following intramuscular injection of 3.12 mM TAT-RasGAP 317-326  (W317A) peptide in the presence or in the absence of 10 mM valinomycin. Survival was visually assessed under a binocular microscope by taking into consideration the embryo transparency (as dead embryos appear opaque), development characteristics and motility.  (G)  Mice were intradermally injected with DiBac4(3) in the presence or in the absence of 10 mM valinomycin. DiBac4(3) fluorescence was then recorded and normalized to the mean of non-treated (NT) control. DiBac4(3) fluorescence was assessed as in panel A.
    Figure Legend Snippet: Related to Fig. 5. Zebrafish and mouse membrane potential modulation. (A) Eighteen hours post fertilization zebrafish embryos were incubated 40 minutes with the indicated concentrations of valinomycin with 950 nM DiBac4(3). DiBac4(3) fluorescence was then recorded and normalized to the non-treated control. The decrease in DiBac4(3) fluorescence indicates membrane hyperpolarization. Membrane potential values could not be calculated, as a standard curve would have to be performed in zebrafish. DiBac4(3) uptake was assessed from confocal images of the fish tail region. (B) Eighteen hours post fertilization zebrafish embryos were incubated with or without 3.12 mM TAT-RasGAP 317-326 (W317A), a mutant version that is not toxic to cells, in the presence of the indicated concentrations of valinomycin. Peptide uptake was assessed from confocal images of the fish tail region. (C-D) Eighteen hours post fertilization zebrafish embryos were incubated 1 hour with the indicated concentrations of valinomycin, in the absence (panel C) or in the presence (panel D) of 3.12 mM TAT-RasGAP 317-326 (W317A), then washed, and incubated in Egg water. The viability of each fish was assessed over 52 hours at the indicated time points. (E) Representative images of zebrafish treated as described in panel C and D, washed, and further incubated in Egg water. Images were taken with a CYTATION3 apparatus at a 4x magnification. (F) Survival of 48 hours post fertilization zebrafish embryos following intramuscular injection of 3.12 mM TAT-RasGAP 317-326 (W317A) peptide in the presence or in the absence of 10 mM valinomycin. Survival was visually assessed under a binocular microscope by taking into consideration the embryo transparency (as dead embryos appear opaque), development characteristics and motility. (G) Mice were intradermally injected with DiBac4(3) in the presence or in the absence of 10 mM valinomycin. DiBac4(3) fluorescence was then recorded and normalized to the mean of non-treated (NT) control. DiBac4(3) fluorescence was assessed as in panel A.

    Techniques Used: Incubation, Fluorescence, Fluorescence In Situ Hybridization, Mutagenesis, Injection, Microscopy, Mouse Assay

    35) Product Images from "Distinct CASK domains control cardiac sodium channel membrane expression and focal adhesion anchoring"

    Article Title: Distinct CASK domains control cardiac sodium channel membrane expression and focal adhesion anchoring

    Journal: bioRxiv

    doi: 10.1101/813030

    CASK interacts with dystrophin through its HOOK domain in cardiomyocytes. (A) Representative western blot of dystrophin expression after transduction with either GFP control, CASK WT , or CASK ΔX . (B) Corresponding histograms showing the expression level of dystrophin normalized to GAPDH in cardiomyocytes transduced with the different adenoviral constructs. Legend: ns, not significant; N=5 independent cell cultures. (C) Representative co-immunoprecipitation assays performed on cardiomyocyte lysates after transduction with CASK WT or CASK ΔX showing the loss of association with dystrophin for the CASK ΔHOOK construct. N=3 independent cell cultures.
    Figure Legend Snippet: CASK interacts with dystrophin through its HOOK domain in cardiomyocytes. (A) Representative western blot of dystrophin expression after transduction with either GFP control, CASK WT , or CASK ΔX . (B) Corresponding histograms showing the expression level of dystrophin normalized to GAPDH in cardiomyocytes transduced with the different adenoviral constructs. Legend: ns, not significant; N=5 independent cell cultures. (C) Representative co-immunoprecipitation assays performed on cardiomyocyte lysates after transduction with CASK WT or CASK ΔX showing the loss of association with dystrophin for the CASK ΔHOOK construct. N=3 independent cell cultures.

    Techniques Used: Western Blot, Expressing, Transduction, Construct, Immunoprecipitation

    Deleting L27B or GUK CASK domains increases cardiac Na V 1.5 channel surface expression. (A) Representatives differential interference contrast (DIC) and Total internal reflection fluorescence microscopy (TIRFm) images of both GFP and Na V 1.5 signal taken from fixed cultured cardiomyocytes transduced with either GFP, CASK WT , CASK ΔL27B , or CASK ΔGUK . (B) Histograms of Na V 1.5 evanescent field fluorescence (EFF) intensity in arbitrary units and normalized to GFP control. Legend: ns, not significant; * P
    Figure Legend Snippet: Deleting L27B or GUK CASK domains increases cardiac Na V 1.5 channel surface expression. (A) Representatives differential interference contrast (DIC) and Total internal reflection fluorescence microscopy (TIRFm) images of both GFP and Na V 1.5 signal taken from fixed cultured cardiomyocytes transduced with either GFP, CASK WT , CASK ΔL27B , or CASK ΔGUK . (B) Histograms of Na V 1.5 evanescent field fluorescence (EFF) intensity in arbitrary units and normalized to GFP control. Legend: ns, not significant; * P

    Techniques Used: Expressing, Fluorescence, Microscopy, Cell Culture, Transduction

    Validation of CASK adenoviral constructs in cardiomyocytes. (A) Schematic representation of CASK structure, domain-specific deletions, and expected molecular weights. (B) Representative western blot showing CASK expression in cultured rat cardiomyocytes transduced with each truncated CASK construct. CASK WT fused to GFP corresponds to a ~140 kDa band. GAPDH served as a loading control. (C) RT-qPCR histograms of CASK mRNA expression levels in cardiomyocytes transduced with GFP, CASK WT , or CASK ΔX , where ΔX corresponds to any deletion. CASK mRNA levels in cardiac cells is normalized to endogenous CASK mRNA levels under control condition (GFP). Legend: ns, not significant; *** P
    Figure Legend Snippet: Validation of CASK adenoviral constructs in cardiomyocytes. (A) Schematic representation of CASK structure, domain-specific deletions, and expected molecular weights. (B) Representative western blot showing CASK expression in cultured rat cardiomyocytes transduced with each truncated CASK construct. CASK WT fused to GFP corresponds to a ~140 kDa band. GAPDH served as a loading control. (C) RT-qPCR histograms of CASK mRNA expression levels in cardiomyocytes transduced with GFP, CASK WT , or CASK ΔX , where ΔX corresponds to any deletion. CASK mRNA levels in cardiac cells is normalized to endogenous CASK mRNA levels under control condition (GFP). Legend: ns, not significant; *** P

    Techniques Used: Construct, Western Blot, Expressing, Cell Culture, Transduction, Quantitative RT-PCR

    Both L27B and GUK CASK domains are implicated in the downregulation of the cardiac sodium current I Na in cultured cardiomyocytes. (A) Current density-voltage relationships (25 mmol/L [Na + ] o ) of I Na obtained from cardiomyocytes transduced with GFP control (black), CASK WT (red), or deleted forms of CASK (CASK ΔX ). (B) Histograms of I Na current density recorded at −20mV and normalized to GFP control. (C-D) Voltage-dependent activation and steady-state inactivation curves from cardiomyocytes transduced with GFP control (black), CASK WT (red), or truncated forms of CASK (CASK ΔX ). Legend: ns, not significant; *** P
    Figure Legend Snippet: Both L27B and GUK CASK domains are implicated in the downregulation of the cardiac sodium current I Na in cultured cardiomyocytes. (A) Current density-voltage relationships (25 mmol/L [Na + ] o ) of I Na obtained from cardiomyocytes transduced with GFP control (black), CASK WT (red), or deleted forms of CASK (CASK ΔX ). (B) Histograms of I Na current density recorded at −20mV and normalized to GFP control. (C-D) Voltage-dependent activation and steady-state inactivation curves from cardiomyocytes transduced with GFP control (black), CASK WT (red), or truncated forms of CASK (CASK ΔX ). Legend: ns, not significant; *** P

    Techniques Used: Cell Culture, Transduction, Activation Assay

    36) Product Images from "Liquid-like protein interactions catalyze assembly of endocytic vesicles"

    Article Title: Liquid-like protein interactions catalyze assembly of endocytic vesicles

    Journal: bioRxiv

    doi: 10.1101/860684

    Eps15 mutants and Fcho1 assemble to varying degrees in solution. ( a-c ) Fcho1 is labeled with Atto-594, Eps15 mutants are labeled with CF488a. Proteins were combined at 7 μM total concentration at physiological pH and salt with 3% PEG. Panels on the left show 7 μM Eps15 mutant alone, set of panels on the right show 6.8 μM Eps15 mutant combined with 0.2 μM Fcho1 (34:1). Cartoons depict binding interaction between Fcho1 and Eps15 mutants. ( a ) Eps15 lacking the EH domains (Eps15-Δ3xEH) does not form droplets on its own, but when combined with Fcho1 forms small droplets. ( b ) Eps15 lacking the C-terminal disordered domain (Eps15-ΔCTD) does not form droplets either on its own or when combined with Fcho1. ( c ) Eps15 containing mutated Fcho1-binding DPF motifs (amino acids 623-636; Eps15-DPF > APA) robustly assembles into droplets on its own and co-assembles into droplets with Fcho1.
    Figure Legend Snippet: Eps15 mutants and Fcho1 assemble to varying degrees in solution. ( a-c ) Fcho1 is labeled with Atto-594, Eps15 mutants are labeled with CF488a. Proteins were combined at 7 μM total concentration at physiological pH and salt with 3% PEG. Panels on the left show 7 μM Eps15 mutant alone, set of panels on the right show 6.8 μM Eps15 mutant combined with 0.2 μM Fcho1 (34:1). Cartoons depict binding interaction between Fcho1 and Eps15 mutants. ( a ) Eps15 lacking the EH domains (Eps15-Δ3xEH) does not form droplets on its own, but when combined with Fcho1 forms small droplets. ( b ) Eps15 lacking the C-terminal disordered domain (Eps15-ΔCTD) does not form droplets either on its own or when combined with Fcho1. ( c ) Eps15 containing mutated Fcho1-binding DPF motifs (amino acids 623-636; Eps15-DPF > APA) robustly assembles into droplets on its own and co-assembles into droplets with Fcho1.

    Techniques Used: Labeling, Concentration Assay, Mutagenesis, Binding Assay

    Eps15 and Fcho1 co-assemble into liquid-like protein droplets. Fcho1 is labeled with Atto-594, Eps15 or Eps15-ΔCC is labeled with CF488a. All droplet experiments are at physiological pH and salt with 3% PEG. ( a ) 7 μM Eps15 forms large, rounded droplets, 7 μM Fcho1 clusters into small, irregular aggregates. Insets show cartoon of inferred protein network assembly within droplets. ( b ) Time course of Eps15-only droplets (upper panels) undergoing fusion (arrowheads) and Fcho1-only aggregates (lower panels) approaching each other but failing to fuse. ( c ) Representative images of fluorescence recovery after bleaching an Eps15-only droplet (upper panels) and an Fcho1-only droplet (lower panels). Plot displays fluorescence recovery curves for each. n=6 experiments, individual data points are shown in gray. Error bars show SEM. ( d ) Phase diagram of Eps15/Fcho1 droplets mapped by CF488a-labeled Eps15 fluorescence intensity. Stars denote critical points for each set of Eps15:Fcho1 ratios. C S and C D indicate the concentration of Eps15 in solution and in droplets, respectively. Horizontal tie lines connect C S and C D for a given temperature. Total protein was held constant at 7 μM. n=3 experiments, error bars show SEM. ( e ) When combined at a 1 to 34 ratio, Fcho1 and Eps15 co-localize in protein droplets. Inset shows cartoon of inferred protein network assembly within droplets, Fcho1 in magenta and Eps15 in green. ( f ) Time course of three fusion events (arrowheads) between droplets containing unlabeled Fcho1 and labeled Eps15. ( g ) Representative images and plot of fluorescence recovery after bleaching a Fcho1 and Eps15 droplet. n=4 experiments, individual data points are shown in gray. Error bars show SEM. ( h ) Time course of protein phase separation induced by the addition of Fcho1 to Eps15. ( i ) At a 1 to 34 ratio, Fcho1 and Eps15-ΔCC do not co-assemble into droplets.
    Figure Legend Snippet: Eps15 and Fcho1 co-assemble into liquid-like protein droplets. Fcho1 is labeled with Atto-594, Eps15 or Eps15-ΔCC is labeled with CF488a. All droplet experiments are at physiological pH and salt with 3% PEG. ( a ) 7 μM Eps15 forms large, rounded droplets, 7 μM Fcho1 clusters into small, irregular aggregates. Insets show cartoon of inferred protein network assembly within droplets. ( b ) Time course of Eps15-only droplets (upper panels) undergoing fusion (arrowheads) and Fcho1-only aggregates (lower panels) approaching each other but failing to fuse. ( c ) Representative images of fluorescence recovery after bleaching an Eps15-only droplet (upper panels) and an Fcho1-only droplet (lower panels). Plot displays fluorescence recovery curves for each. n=6 experiments, individual data points are shown in gray. Error bars show SEM. ( d ) Phase diagram of Eps15/Fcho1 droplets mapped by CF488a-labeled Eps15 fluorescence intensity. Stars denote critical points for each set of Eps15:Fcho1 ratios. C S and C D indicate the concentration of Eps15 in solution and in droplets, respectively. Horizontal tie lines connect C S and C D for a given temperature. Total protein was held constant at 7 μM. n=3 experiments, error bars show SEM. ( e ) When combined at a 1 to 34 ratio, Fcho1 and Eps15 co-localize in protein droplets. Inset shows cartoon of inferred protein network assembly within droplets, Fcho1 in magenta and Eps15 in green. ( f ) Time course of three fusion events (arrowheads) between droplets containing unlabeled Fcho1 and labeled Eps15. ( g ) Representative images and plot of fluorescence recovery after bleaching a Fcho1 and Eps15 droplet. n=4 experiments, individual data points are shown in gray. Error bars show SEM. ( h ) Time course of protein phase separation induced by the addition of Fcho1 to Eps15. ( i ) At a 1 to 34 ratio, Fcho1 and Eps15-ΔCC do not co-assemble into droplets.

    Techniques Used: Labeling, Fluorescence, Concentration Assay

    Eps15 and Fcho1 assemble into protein-rich domains on membrane surfaces. ( a-e ) Center slices (upper panels) and corresponding z-projections (lower panels) of representative GUVs incubated with 500 nM of the indicated protein(s). Fcho1 is labeled with Atto-594 and Eps15 variants are labeled with CF488a. GUVs contain 79% DOPC, 15% DOPS, 5% PtdIns(4,5)P 2 , and 1% DPEG10-biotin unless otherwise indicated. Scale bars are 5 μm. ( a ) Full-length Fcho1 alone on GUVs, (left) and full-length Eps15 alone on GUVs containing 97% DOPC, 2% DOGS-NTA-Ni, 1% DPEG10-biotin (center). Cartoons (right) depict domain organization of Fcho1 and Eps15 dimeric forms. ( b ) GUVs incubated with both Fcho1 and Eps15 display several protein-rich domains or ( c ) a single protein-rich domain. ( d ) GUVs labeled with 0.1% Texas Red-DHPE lipid were incubated with 500 nM each of CF488a-labeled Eps15 and unlabeled Fcho1. ( e ) GUVs incubated with Fcho1 and Eps15 lacking the coiled-coil domain (Eps15-ΔCC) do not display protein-rich domains. Cartoon depicts interaction between Fcho1 and an Eps15-ΔCC monomer. ( f ) Frequency of GUVs displaying protein-rich domains for each set of proteins. For each bar, n=4 experiments with at least 50 total GUVs for each condition, error bars show SEM. ( g-i ) Multibilayers containing 73% DOPC, 25% DOPS, and 2% DOGS-NTA-Ni were incubated with 100 nM Atto594-labeled Fcho1, or 100 nM CF488a-labeled Eps15, or both. Scale bar is 5 μm. (g) Fcho1 and Eps15 individually decorate multibilayers homogeneously. When combined, Fcho1 and Eps15 form micron-scale protein-rich regions. Scale bar is 5 μm. ( h ) Time course of protein-rich Eps15/Fcho1 domains merging on a multibilayer, 6 s intervals. Scale bar is 3 μm. (i) Representative images and plot of fluorescence recovery after bleaching Eps15 (green)/Fcho1 (unlabeled) protein domains on multibilayers. n=3 experiments, individual data points are shown in gray. Error bars show SEM.
    Figure Legend Snippet: Eps15 and Fcho1 assemble into protein-rich domains on membrane surfaces. ( a-e ) Center slices (upper panels) and corresponding z-projections (lower panels) of representative GUVs incubated with 500 nM of the indicated protein(s). Fcho1 is labeled with Atto-594 and Eps15 variants are labeled with CF488a. GUVs contain 79% DOPC, 15% DOPS, 5% PtdIns(4,5)P 2 , and 1% DPEG10-biotin unless otherwise indicated. Scale bars are 5 μm. ( a ) Full-length Fcho1 alone on GUVs, (left) and full-length Eps15 alone on GUVs containing 97% DOPC, 2% DOGS-NTA-Ni, 1% DPEG10-biotin (center). Cartoons (right) depict domain organization of Fcho1 and Eps15 dimeric forms. ( b ) GUVs incubated with both Fcho1 and Eps15 display several protein-rich domains or ( c ) a single protein-rich domain. ( d ) GUVs labeled with 0.1% Texas Red-DHPE lipid were incubated with 500 nM each of CF488a-labeled Eps15 and unlabeled Fcho1. ( e ) GUVs incubated with Fcho1 and Eps15 lacking the coiled-coil domain (Eps15-ΔCC) do not display protein-rich domains. Cartoon depicts interaction between Fcho1 and an Eps15-ΔCC monomer. ( f ) Frequency of GUVs displaying protein-rich domains for each set of proteins. For each bar, n=4 experiments with at least 50 total GUVs for each condition, error bars show SEM. ( g-i ) Multibilayers containing 73% DOPC, 25% DOPS, and 2% DOGS-NTA-Ni were incubated with 100 nM Atto594-labeled Fcho1, or 100 nM CF488a-labeled Eps15, or both. Scale bar is 5 μm. (g) Fcho1 and Eps15 individually decorate multibilayers homogeneously. When combined, Fcho1 and Eps15 form micron-scale protein-rich regions. Scale bar is 5 μm. ( h ) Time course of protein-rich Eps15/Fcho1 domains merging on a multibilayer, 6 s intervals. Scale bar is 3 μm. (i) Representative images and plot of fluorescence recovery after bleaching Eps15 (green)/Fcho1 (unlabeled) protein domains on multibilayers. n=3 experiments, individual data points are shown in gray. Error bars show SEM.

    Techniques Used: Incubation, Labeling, Fluorescence

    37) Product Images from "Glial cell mechanosensitivity is reversed by adhesion cues"

    Article Title: Glial cell mechanosensitivity is reversed by adhesion cues

    Journal: bioRxiv

    doi: 10.1101/865303

    Glial cell initial adhesion and proliferation on poly-L-lysine/laminin coated substrates in DMEMs culture medium. A - Glial cells from pure culture at the stiff (42 kPa) - soft (1.1 kPa) frontier on a “concentric” pattern of rigidity at 17 DIV, and the evolution of the cell density over time (days in vitro, DIV) on stiff (black squares) and soft (white circles) regions. ** denotes that the two means are significantly different, p (stiff vs. soft) = 0.0016 (2 DIV); ****, p
    Figure Legend Snippet: Glial cell initial adhesion and proliferation on poly-L-lysine/laminin coated substrates in DMEMs culture medium. A - Glial cells from pure culture at the stiff (42 kPa) - soft (1.1 kPa) frontier on a “concentric” pattern of rigidity at 17 DIV, and the evolution of the cell density over time (days in vitro, DIV) on stiff (black squares) and soft (white circles) regions. ** denotes that the two means are significantly different, p (stiff vs. soft) = 0.0016 (2 DIV); ****, p

    Techniques Used: In Vitro

    Rigidity sensitivity in DMEMs of glial cells from mixed cultures is enhanced compared to pure cultures. A - Comparison of the normalized initial adhesion of glial cells from pure cultures on the different coatings (“FN”: fibronectin, “PLL/LN”: poly-L-lysine/laminin) and rigidities (“Soft”: 1.1 kPa, “Stiff”: 42 kPa). B - Normalized initial proliferation rates show that glial cells proliferate faster in mixed culture conditions. C - Comparison of the evolution of the cell density in pure and mixed cultures on the stiff regions coated with FN.
    Figure Legend Snippet: Rigidity sensitivity in DMEMs of glial cells from mixed cultures is enhanced compared to pure cultures. A - Comparison of the normalized initial adhesion of glial cells from pure cultures on the different coatings (“FN”: fibronectin, “PLL/LN”: poly-L-lysine/laminin) and rigidities (“Soft”: 1.1 kPa, “Stiff”: 42 kPa). B - Normalized initial proliferation rates show that glial cells proliferate faster in mixed culture conditions. C - Comparison of the evolution of the cell density in pure and mixed cultures on the stiff regions coated with FN.

    Techniques Used:

    Neuronal cell growth in mixed cultures on laminin/Poly-L-lysine coated substrates in NBs culture medium. A, B - Phase contrast imaging of neuronal cells on a soft (A, 1.1 kPa) and high stiffness (B, 42 kPa) on “concentric” pattern of rigidity at 14 days in vitro (DIV). Scale bars: 50 µm. C - Neuron density and its evolution over time (DIV) on stiff (black squares) and soft (white circles) regions. The blue dot indicates the initial cell concentration (including both glial and neuronal cells) seeded on the substrate. Quantification of the cell density is presented as mean ± standard deviation (SD). * denotes that the two means are significantly different, p (stiff vs. soft at 3 DIV) = 0.0380. Slopes not statistically different from zero: p (soft) = 0.1502; p (stiff) = 0.6781. The grey and black lines in the graphes represent the y-mean value of the linear fit. D - Phase contrast imaging of neurons at the frontier of between the low and the high stiffness regions on the “double rigidity” design. E - Neurite orientation at the frontier between the soft and the stiff regions, characterized by the angle α perpendicular to the frontier as shown in D. The histogram for α is centered at 0 deg.
    Figure Legend Snippet: Neuronal cell growth in mixed cultures on laminin/Poly-L-lysine coated substrates in NBs culture medium. A, B - Phase contrast imaging of neuronal cells on a soft (A, 1.1 kPa) and high stiffness (B, 42 kPa) on “concentric” pattern of rigidity at 14 days in vitro (DIV). Scale bars: 50 µm. C - Neuron density and its evolution over time (DIV) on stiff (black squares) and soft (white circles) regions. The blue dot indicates the initial cell concentration (including both glial and neuronal cells) seeded on the substrate. Quantification of the cell density is presented as mean ± standard deviation (SD). * denotes that the two means are significantly different, p (stiff vs. soft at 3 DIV) = 0.0380. Slopes not statistically different from zero: p (soft) = 0.1502; p (stiff) = 0.6781. The grey and black lines in the graphes represent the y-mean value of the linear fit. D - Phase contrast imaging of neurons at the frontier of between the low and the high stiffness regions on the “double rigidity” design. E - Neurite orientation at the frontier between the soft and the stiff regions, characterized by the angle α perpendicular to the frontier as shown in D. The histogram for α is centered at 0 deg.

    Techniques Used: Imaging, In Vitro, Concentration Assay, Standard Deviation

    38) Product Images from "Towards the Fecal Metabolome Derived from Moderate Red Wine Intake"

    Article Title: Towards the Fecal Metabolome Derived from Moderate Red Wine Intake

    Journal: Metabolites

    doi: 10.3390/metabo4041101

    Overlapped MRM chromatograms before ( a ) and after ( b ) wine intake obtained for Volunteer 1. (1) Gallic acid; (2) 3,5-dihydroxybenzoic acid; (3) 3-O-methylgallic acid; (4) 3,4-dihydroxyphenylacetic acid; (5) protocatechuic acid; (6) 4-hydroxybenzoic acid; (7) 4-hydroxy-5-(3’,4’-dihydroxyphenyl)-valeric acid; (8) vanillic acid; (9) 3-hydroxyphenylacetic acid; (10) 3-(4-hydroxyphenyl)-propionic acid; (11) syringic acid; (12) 5-(3’,4’-dihydroxyphenyl)-γ-valerolactone; (13) p -coumaric acid; (14) 4-hydroxy-5-(3’-hydroxyphenyl)-valeric acid; (15) 3-(3-hydroxyphenyl)-propionic acid; (16) ferulic acid; (17) benzoic acid; (18) phenylacetic acid; (19) salicylic acid; (20) 5-(3’-hydroxyphenyl)-γ-valerolactone; (21) phenylpropionic acid; and (22) 4-hydroxy-5-(phenyl)-valeric acid.
    Figure Legend Snippet: Overlapped MRM chromatograms before ( a ) and after ( b ) wine intake obtained for Volunteer 1. (1) Gallic acid; (2) 3,5-dihydroxybenzoic acid; (3) 3-O-methylgallic acid; (4) 3,4-dihydroxyphenylacetic acid; (5) protocatechuic acid; (6) 4-hydroxybenzoic acid; (7) 4-hydroxy-5-(3’,4’-dihydroxyphenyl)-valeric acid; (8) vanillic acid; (9) 3-hydroxyphenylacetic acid; (10) 3-(4-hydroxyphenyl)-propionic acid; (11) syringic acid; (12) 5-(3’,4’-dihydroxyphenyl)-γ-valerolactone; (13) p -coumaric acid; (14) 4-hydroxy-5-(3’-hydroxyphenyl)-valeric acid; (15) 3-(3-hydroxyphenyl)-propionic acid; (16) ferulic acid; (17) benzoic acid; (18) phenylacetic acid; (19) salicylic acid; (20) 5-(3’-hydroxyphenyl)-γ-valerolactone; (21) phenylpropionic acid; and (22) 4-hydroxy-5-(phenyl)-valeric acid.

    Techniques Used:

    39) Product Images from "Neural Epidermal Growth Factor-Like Like Protein 2 (NELL2) Promotes Aggregation of Embryonic Carcinoma P19 Cells by Inducing N-Cadherin Expression"

    Article Title: Neural Epidermal Growth Factor-Like Like Protein 2 (NELL2) Promotes Aggregation of Embryonic Carcinoma P19 Cells by Inducing N-Cadherin Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085898

    Retinoic acid (RA) activates m NELL2 promoter activity and expression of endogenous NELL2 mRNA and protein in P19 cells. (A) To determine the effects of RA on m NELL2 promoter activity, a 1.3 kb m NELL2 promoter-luciferase (luc) reporter construct was transfected into the P19 cells, and the luciferase activity was determined 24 h after the RA treatment. (B) Real-time PCR analysis showing the change in endogenous NELL2 mRNA expression by RA treatment. (C) Western blot analysis to determine changes in RA-induced NELL2 expression. High concentration (1 µM) of RA increased intracellular NELL2 or secreted NELL2 (sNELL2). All experiments were repeated at least four times and data are presented as mean ± SEM. *, p
    Figure Legend Snippet: Retinoic acid (RA) activates m NELL2 promoter activity and expression of endogenous NELL2 mRNA and protein in P19 cells. (A) To determine the effects of RA on m NELL2 promoter activity, a 1.3 kb m NELL2 promoter-luciferase (luc) reporter construct was transfected into the P19 cells, and the luciferase activity was determined 24 h after the RA treatment. (B) Real-time PCR analysis showing the change in endogenous NELL2 mRNA expression by RA treatment. (C) Western blot analysis to determine changes in RA-induced NELL2 expression. High concentration (1 µM) of RA increased intracellular NELL2 or secreted NELL2 (sNELL2). All experiments were repeated at least four times and data are presented as mean ± SEM. *, p

    Techniques Used: Activity Assay, Expressing, Luciferase, Construct, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Concentration Assay

    EMSA and ChIP assays. EMSAs were performed using double-stranded oligomer probes containing the putative half-RAREs found in the m NELL2 promoter sequence. (A) Autoradiogram showing binding activity of half-RAREs derived from the m NELL2 promoter to the nuclear extracts from P19 cells treated with RA (1 µM). Numbers on the gel images indicate the sites in the m NELL2 promoter sequence where the oligomer probes were designed; M following the numbers means that the indicted probes bear a mutation in the half-RARE sites. B, protein-bound DNA; F, free DNA; PC, a positive control palindromic RARE; NC, a negative control of mutant RARE. (B) ChIP assays using DNA precipitated by using RAR antibodies. The immunoprecipitated DNA from P19 cells treated with RA or DMSO was PCR-amplified using primer sets designed to detect m NELL2 promoter fragments including the two half-RARE sequences (at −223 and −1047). Input represents the used DNA extracted from the P19 cells before immunoprecipitation. Normal rabbit IgG was included for immunoprecipitation in the assay as a negative control.
    Figure Legend Snippet: EMSA and ChIP assays. EMSAs were performed using double-stranded oligomer probes containing the putative half-RAREs found in the m NELL2 promoter sequence. (A) Autoradiogram showing binding activity of half-RAREs derived from the m NELL2 promoter to the nuclear extracts from P19 cells treated with RA (1 µM). Numbers on the gel images indicate the sites in the m NELL2 promoter sequence where the oligomer probes were designed; M following the numbers means that the indicted probes bear a mutation in the half-RARE sites. B, protein-bound DNA; F, free DNA; PC, a positive control palindromic RARE; NC, a negative control of mutant RARE. (B) ChIP assays using DNA precipitated by using RAR antibodies. The immunoprecipitated DNA from P19 cells treated with RA or DMSO was PCR-amplified using primer sets designed to detect m NELL2 promoter fragments including the two half-RARE sequences (at −223 and −1047). Input represents the used DNA extracted from the P19 cells before immunoprecipitation. Normal rabbit IgG was included for immunoprecipitation in the assay as a negative control.

    Techniques Used: Chromatin Immunoprecipitation, Sequencing, Binding Assay, Activity Assay, Derivative Assay, Mutagenesis, Positive Control, Negative Control, Immunoprecipitation, Polymerase Chain Reaction, Amplification

    Change in NELL2 expression during the RA-induced neuronal differentiation of P19 cells. (A) General scheme for the neuronal differentiation process in this study. P19 cells were aggregated for 4 days with 1 µM RA treatment, and the aggregates were harvested and replated as the single-cell suspension and cultured without RA for 4 days. (B) Representative photos showing morphological changes in P19 cells during the neuronal differentiation process. Cells show aggregated morphologies as embryonic bodies at 2 and 4 days of aggregation, and reveal bipolar shapes with processes at 2 and 4 days after replating. Control (CTL) represents 2 days of aggregation without any treatment. (C, E, F) Real-time PCR analysis of NELL2 (C), Tuj-1 (E) and Nestin (F) mRNA expression in the P19 cells during the process of aggregation and replating. Agg, aggregation; Rep, replating; AU, arbitrary units. All experiments were repeated at least four times and data are presented as mean ± SEM. *, p
    Figure Legend Snippet: Change in NELL2 expression during the RA-induced neuronal differentiation of P19 cells. (A) General scheme for the neuronal differentiation process in this study. P19 cells were aggregated for 4 days with 1 µM RA treatment, and the aggregates were harvested and replated as the single-cell suspension and cultured without RA for 4 days. (B) Representative photos showing morphological changes in P19 cells during the neuronal differentiation process. Cells show aggregated morphologies as embryonic bodies at 2 and 4 days of aggregation, and reveal bipolar shapes with processes at 2 and 4 days after replating. Control (CTL) represents 2 days of aggregation without any treatment. (C, E, F) Real-time PCR analysis of NELL2 (C), Tuj-1 (E) and Nestin (F) mRNA expression in the P19 cells during the process of aggregation and replating. Agg, aggregation; Rep, replating; AU, arbitrary units. All experiments were repeated at least four times and data are presented as mean ± SEM. *, p

    Techniques Used: Expressing, Cell Culture, CTL Assay, Real-time Polymerase Chain Reaction

    NELL2 promotes neuronal differentiation of P19 cells. (A) Overexpression of NELL2 in P19 cells permanently transfected with NELL2 expression vectors confirmed by RT-PCR using RNA extracted from the cells. CTL, control cells; pcDNA, cells transfected with control pcDNA vectors; NELL2, cells transfected with NELL2 expression vectors. (B). Western blot analysis for overexpression of NELL2 protein in the P19 cells permanently transfected with NELL2 expression vectors. (C) Representative photograms showing the morphological changes of P19 cells by overexpression of the NELL2 expression vectors with or without the treatment of RA (1 µM). (D–F) Real-time PCR analysis of Ngn-1 (D), Nestin (E) and Tuj-1 (F) mRNA expression in the P19 cells overexpressing NELL2 during the aggregation and replating process of the neuronal differentiation. RNA samples were collected from P19 cells with the indicated treatment at 2 and 4 days after aggregation and replating. (G) Western blot analysis of NeuN protein expression in P19 cells during aggregation and replating processes. All experiments were repeated at least four times and data are presented as mean ± SEM. *, p
    Figure Legend Snippet: NELL2 promotes neuronal differentiation of P19 cells. (A) Overexpression of NELL2 in P19 cells permanently transfected with NELL2 expression vectors confirmed by RT-PCR using RNA extracted from the cells. CTL, control cells; pcDNA, cells transfected with control pcDNA vectors; NELL2, cells transfected with NELL2 expression vectors. (B). Western blot analysis for overexpression of NELL2 protein in the P19 cells permanently transfected with NELL2 expression vectors. (C) Representative photograms showing the morphological changes of P19 cells by overexpression of the NELL2 expression vectors with or without the treatment of RA (1 µM). (D–F) Real-time PCR analysis of Ngn-1 (D), Nestin (E) and Tuj-1 (F) mRNA expression in the P19 cells overexpressing NELL2 during the aggregation and replating process of the neuronal differentiation. RNA samples were collected from P19 cells with the indicated treatment at 2 and 4 days after aggregation and replating. (G) Western blot analysis of NeuN protein expression in P19 cells during aggregation and replating processes. All experiments were repeated at least four times and data are presented as mean ± SEM. *, p

    Techniques Used: Over Expression, Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, CTL Assay, Western Blot, Real-time Polymerase Chain Reaction

    Effect of NELL2 on the N-cadherin expression in P19 cells. (A, B) Real-time PCR analysis of E-cadherin (A) and N-cadherin (B) mRNA expression in the P19 cells expressing NELL2 with or without treatment of RA, as indicated. For real-time PCR analysis, RNA samples were harvested from the cells at 2 and 4 days after aggregation and replating. (C) Data showing changes in N-cadherin protein expression calculated from Western blot analysis of samples collected at the aggregation and replating processes of neuronal differentiation of P19 cells. All experiments were repeated at least four times and data are presented as mean ± SEM. *, p
    Figure Legend Snippet: Effect of NELL2 on the N-cadherin expression in P19 cells. (A, B) Real-time PCR analysis of E-cadherin (A) and N-cadherin (B) mRNA expression in the P19 cells expressing NELL2 with or without treatment of RA, as indicated. For real-time PCR analysis, RNA samples were harvested from the cells at 2 and 4 days after aggregation and replating. (C) Data showing changes in N-cadherin protein expression calculated from Western blot analysis of samples collected at the aggregation and replating processes of neuronal differentiation of P19 cells. All experiments were repeated at least four times and data are presented as mean ± SEM. *, p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    NELL2 regulates N-cadherin expression through the ERK signaling. P19 cells permanently transfected with NELL2 expression vectors were treated with 5 µM U0126 (a MEK inhibitor) in the presence or absence of 1 µM RA for 4 days. (A) Representative Western blots showing effect of U0126 on the ERK phosphorylation and N-cadherin and c-Fos expression. (B–D) Data showing the U0126-induced changes in phosphorylation of ERK (B) and expression of N-cadherin (C) and c-Fos (D), calculated from Western blot analyses. All experiments were repeated at least four times and data are presented as mean ± SEM. *, p
    Figure Legend Snippet: NELL2 regulates N-cadherin expression through the ERK signaling. P19 cells permanently transfected with NELL2 expression vectors were treated with 5 µM U0126 (a MEK inhibitor) in the presence or absence of 1 µM RA for 4 days. (A) Representative Western blots showing effect of U0126 on the ERK phosphorylation and N-cadherin and c-Fos expression. (B–D) Data showing the U0126-induced changes in phosphorylation of ERK (B) and expression of N-cadherin (C) and c-Fos (D), calculated from Western blot analyses. All experiments were repeated at least four times and data are presented as mean ± SEM. *, p

    Techniques Used: Expressing, Transfection, Western Blot

    NELL2 promotes aggregation of P19 cells. P19 cells permanently transfected with NELL2 expression vectors were cultured in the presence or absence of 1 µM RA for 2 (A) or 4 (B) days. To knock down NELL2 synthesis, the indicated groups of P19 cells were transfected with siRNA against NELL2 mRNA (siNELL2). Each upper panel reveals representative photos and the lower panel includes results showing difference in single cell numbers among treatment groups. All experiments were repeated at least four times and data are presented as mean ± SEM. *, p
    Figure Legend Snippet: NELL2 promotes aggregation of P19 cells. P19 cells permanently transfected with NELL2 expression vectors were cultured in the presence or absence of 1 µM RA for 2 (A) or 4 (B) days. To knock down NELL2 synthesis, the indicated groups of P19 cells were transfected with siRNA against NELL2 mRNA (siNELL2). Each upper panel reveals representative photos and the lower panel includes results showing difference in single cell numbers among treatment groups. All experiments were repeated at least four times and data are presented as mean ± SEM. *, p

    Techniques Used: Transfection, Expressing, Cell Culture

    A hypothetical model for NELL2 action in the neuronal differentiation of P19 cells. RA diffuses across the plasma membrane and RA-receptor (RAR/RXR) complexes bind to the RA response elements (RAREs) of the NELL2 promoter region. These complexes transcriptionally activate NELL2 expression, and, in turn, released NELL2 stimulates the ERK pathway via as yet unknown receptor signaling. The activated ERK increases synthesis of N-cadherin, which mediates cell-cell adhesion and neuronal differentiation.
    Figure Legend Snippet: A hypothetical model for NELL2 action in the neuronal differentiation of P19 cells. RA diffuses across the plasma membrane and RA-receptor (RAR/RXR) complexes bind to the RA response elements (RAREs) of the NELL2 promoter region. These complexes transcriptionally activate NELL2 expression, and, in turn, released NELL2 stimulates the ERK pathway via as yet unknown receptor signaling. The activated ERK increases synthesis of N-cadherin, which mediates cell-cell adhesion and neuronal differentiation.

    Techniques Used: Expressing

    40) Product Images from "Angiopoietin1 Inhibits Mast Cell Activation and Protects against Anaphylaxis"

    Article Title: Angiopoietin1 Inhibits Mast Cell Activation and Protects against Anaphylaxis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0089148

    Ang-1 inhibited LPS-induced cytokines production in P815 mast cells. Quantitative RT-PCR (A and B) and ELISA (C and D) were employed for detection of mRNA and the secretion of cytokines production in duplicates, respectively. A and C: LPS dramatically increased TNF-α mRNA production and protein secretion in P815 mast cells (column 2 versus column 1). Addition of Ang-1 abrogated the induction of LPS on mast cells (column 3 versus column 2). Soluble form of Tie2 (sTie-2) and RGD reversed the inhibition of Ang-1 on LPS-induced TNF-α production (column 4, 5 versus column 3). *P
    Figure Legend Snippet: Ang-1 inhibited LPS-induced cytokines production in P815 mast cells. Quantitative RT-PCR (A and B) and ELISA (C and D) were employed for detection of mRNA and the secretion of cytokines production in duplicates, respectively. A and C: LPS dramatically increased TNF-α mRNA production and protein secretion in P815 mast cells (column 2 versus column 1). Addition of Ang-1 abrogated the induction of LPS on mast cells (column 3 versus column 2). Soluble form of Tie2 (sTie-2) and RGD reversed the inhibition of Ang-1 on LPS-induced TNF-α production (column 4, 5 versus column 3). *P

    Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Inhibition

    Ang-1 inhibited LPS-induced IκB phosphorylation and NF-κB nuclear translocation in P815 mast cells. A: Immunofluorescence showed Tie-2 receptor expression. Primary antibody in negative control was monoclonal IgG. B: Western blotting was performed to analyze phosphorylation levels of IκB in mast cells in response to different stimuli. C and D: Densitometric analysis was used to calculate the relative ratio of p-IκB/β-actin (C) and IκB/β-actin (D). Ratio of the control group was arbitrarily presented as 1. *P
    Figure Legend Snippet: Ang-1 inhibited LPS-induced IκB phosphorylation and NF-κB nuclear translocation in P815 mast cells. A: Immunofluorescence showed Tie-2 receptor expression. Primary antibody in negative control was monoclonal IgG. B: Western blotting was performed to analyze phosphorylation levels of IκB in mast cells in response to different stimuli. C and D: Densitometric analysis was used to calculate the relative ratio of p-IκB/β-actin (C) and IκB/β-actin (D). Ratio of the control group was arbitrarily presented as 1. *P

    Techniques Used: Translocation Assay, Immunofluorescence, Expressing, Negative Control, Western Blot

    Ang-1 suppressed compound 48/80 induced mast cells degranulation. Degranulation was determined by staining with dyes and measuring the release of histamine and tryptase. A: Mast cells degranulation was observed by microscope 20 min after compound 48/80 10 µg/mL treatment. Cells were stained with alcian blue (a–e) and toluidine blue (f–j) (100×). (a, f) control group; (b, g) compound 48/80-treated cells; (c, h) Ang-1 100 ng/ml-treated cells; (d, i) Soluble form of Tie2 (sTie-2)-treated cells and (e, j) RGD-treated cells. B and C: Quantification of P815 mast cells degranulation by compound 48/80. It was performed in a blinded fashion. D: Degranulation stimulated by compound 48/80 was determined by measuring the release of tryptaseβ2 (mMCP-6) through commercial ELISA kit in duplicates. The data shown are mean±SD of 3 separate experiments. E: Degranulation stimulated by compound 48/80 was determined by measuring the release of histamine through OPT-fluorometric assay as previously reported in duplicates. F: Mast cells degranulation were incubated with 250 ng/ml before DNP-BSA 10 µg/ml treatment for 20 min. Degranulation was determined by measuring the release of tryptase-β2 (mMCP-6) through commercial ELISA kit in duplicates. The data shown are mean ±SD of 3 separate experiments. G: Mast cells degranulation were incubated with 250 ng/ml before DNP-BSA 10 µg/ml treatment for 20 min. Degranulation was determined by measuring the release of histamine through OPT-fluorometric assay as previously reported in duplicates. *P
    Figure Legend Snippet: Ang-1 suppressed compound 48/80 induced mast cells degranulation. Degranulation was determined by staining with dyes and measuring the release of histamine and tryptase. A: Mast cells degranulation was observed by microscope 20 min after compound 48/80 10 µg/mL treatment. Cells were stained with alcian blue (a–e) and toluidine blue (f–j) (100×). (a, f) control group; (b, g) compound 48/80-treated cells; (c, h) Ang-1 100 ng/ml-treated cells; (d, i) Soluble form of Tie2 (sTie-2)-treated cells and (e, j) RGD-treated cells. B and C: Quantification of P815 mast cells degranulation by compound 48/80. It was performed in a blinded fashion. D: Degranulation stimulated by compound 48/80 was determined by measuring the release of tryptaseβ2 (mMCP-6) through commercial ELISA kit in duplicates. The data shown are mean±SD of 3 separate experiments. E: Degranulation stimulated by compound 48/80 was determined by measuring the release of histamine through OPT-fluorometric assay as previously reported in duplicates. F: Mast cells degranulation were incubated with 250 ng/ml before DNP-BSA 10 µg/ml treatment for 20 min. Degranulation was determined by measuring the release of tryptase-β2 (mMCP-6) through commercial ELISA kit in duplicates. The data shown are mean ±SD of 3 separate experiments. G: Mast cells degranulation were incubated with 250 ng/ml before DNP-BSA 10 µg/ml treatment for 20 min. Degranulation was determined by measuring the release of histamine through OPT-fluorometric assay as previously reported in duplicates. *P

    Techniques Used: Staining, Microscopy, Enzyme-linked Immunosorbent Assay, Incubation

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    Transferring:

    Article Title: Isolation and characterization of GFAP-positive porcine neural stem/progenitor cells derived from a GFAP-CreERT2 transgenic piglet
    Article Snippet: .. Differentiation of pGFAP-CreERT2 -NSCs For spontaneous NSC differentiation, neurospheres were seeded in wells containing poly-d -lysine (Sigma), using a Pasteur pipette, and cultured in the absence of EGF and bFGF for 10 days. .. To differentiate NSCs into astrocytes, after reaching 70–80% confluency, the NSC culture medium was replaced with astrocyte differentiation medium consisting of N2B27 media supplemented with 1% FBS (Gibco).

    Cell Culture:

    Article Title: Isolation and characterization of GFAP-positive porcine neural stem/progenitor cells derived from a GFAP-CreERT2 transgenic piglet
    Article Snippet: .. Differentiation of pGFAP-CreERT2 -NSCs For spontaneous NSC differentiation, neurospheres were seeded in wells containing poly-d -lysine (Sigma), using a Pasteur pipette, and cultured in the absence of EGF and bFGF for 10 days. .. To differentiate NSCs into astrocytes, after reaching 70–80% confluency, the NSC culture medium was replaced with astrocyte differentiation medium consisting of N2B27 media supplemented with 1% FBS (Gibco).

    Article Title: Possible Insulinotropic Action of Apolipoprotein A–I Through the ABCA1/Cdc42/cAMP/PKA Pathway in MIN6 Cells
    Article Snippet: .. The effect of glucose (final concentration 0.5, 5.5, and 25 mmol/l) on cAMP production was determined in MIN6 cells cultured in the DMEM medium with 50 μg/ml of ApoA-I (apolipoprotein A-I from human plasma; Sigma-Aldrich, St. Louis, MO, USA). .. Then, the effect of ApoA-I (final concentration 10, 25, and 50 ug/ml) on cAMP production was determined in the medium with 25 mmol/l of glucose.

    Concentration Assay:

    Article Title: Possible Insulinotropic Action of Apolipoprotein A–I Through the ABCA1/Cdc42/cAMP/PKA Pathway in MIN6 Cells
    Article Snippet: .. The effect of glucose (final concentration 0.5, 5.5, and 25 mmol/l) on cAMP production was determined in MIN6 cells cultured in the DMEM medium with 50 μg/ml of ApoA-I (apolipoprotein A-I from human plasma; Sigma-Aldrich, St. Louis, MO, USA). .. Then, the effect of ApoA-I (final concentration 10, 25, and 50 ug/ml) on cAMP production was determined in the medium with 25 mmol/l of glucose.

    Incubation:

    Article Title: Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells
    Article Snippet: .. Cells were permeabilized with 0.1% Triton X-100 in PBS, blocked with 5% horse serum in PBS, and incubated with Ndr2 rabbit Abs and Cy3-labeled CD3 mAb clone 145-2C11) or Ndr2 rabbit Abs in combination with TRITC-conjugated phalloidin (Sigma Aldrich). .. Bound Ndr2 antibodies were detected with FITC-conjugated goat anti-rabbit IgG (Dianova).

    Modification:

    Article Title: Chemogenetic Approach Using Ni(II) Complex–Agonist Conjugates Allows Selective Activation of Class A G-Protein-Coupled Receptors
    Article Snippet: .. Culture and Transfection of HEK293T, HEK293, and CHO Cells HEK293T, HEK293, and CHO cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) for HEK293T and HEK293 cells or DMEM-F12 (Sigma-Aldrich) for CHO cells supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 unit/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (Gibco) at 37 °C in a humidified atmosphere of 95% air and 5% CO2 . .. For β2 ARs, HEK293T and HEK293 cells were transiently transfected with plasmids (WT β2 ARs, the β2 AR mutants, or the control vector) using Lipofectamine2000 (Invitrogen) and Superfect transfection reagent (Qiagen), respectively, in DMEM supplemented with 10% FBS according to the manufacture’s instruction.

    Western Blot:

    Article Title: The mechanism of miR-142-3p in coronary microembolization-induced myocardiac injury via regulating target gene IRAK-1
    Article Snippet: .. Western blot analysis Total proteins obtained from the cardiac tissues and cardiomyocytes were separated by 10–15% SDS–PAGE and then electrotransferred onto PVDF membranes (Millipore, Atlanta, GA, US). .. The membranes were blocked with 5% bovine serum albumin or non-fat milk for 1.5 h at room temperature, followed by incubation at 4 °C overnight with primary antibodies against IRAK-1, NF-κB p65, TNF-α, IL-1β, IL-6, or GAPDH.

    SDS Page:

    Article Title: The mechanism of miR-142-3p in coronary microembolization-induced myocardiac injury via regulating target gene IRAK-1
    Article Snippet: .. Western blot analysis Total proteins obtained from the cardiac tissues and cardiomyocytes were separated by 10–15% SDS–PAGE and then electrotransferred onto PVDF membranes (Millipore, Atlanta, GA, US). .. The membranes were blocked with 5% bovine serum albumin or non-fat milk for 1.5 h at room temperature, followed by incubation at 4 °C overnight with primary antibodies against IRAK-1, NF-κB p65, TNF-α, IL-1β, IL-6, or GAPDH.

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  • 99
    Millipore anti mono ubiquitinated histone h2a lysine 119
    Dramatically decreased mono-ubiquitination of histone <t>H2A</t> <t>lysine</t> 119 (H2AK119u1) and phosphorylation of enhancer of zeste homolog 2 (Ezh2) methyltransferase in parthenogenetic blastocysts. Immunostaining was used to reveal that levels of total Ezh2 methyltransferase
    Anti Mono Ubiquitinated Histone H2a Lysine 119, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mono ubiquitinated histone h2a lysine 119/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti mono ubiquitinated histone h2a lysine 119 - by Bioz Stars, 2020-09
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    91
    Millipore lysine 9
    Comparison of global histone modification patterns of KSHV genomes in BrK.219 and PEL cells. Global patterns of histone H3 trimethylated at lysine 4 (H3K4-me3), <t>lysine</t> 9 (H3K9-me3), or lysine 27 (H3K27-me3) or acetylated at lysine 9 and/or lysine 14 (H3K27-me3
    Lysine 9, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore lysine 4
    Chromatin immunoprecipitation using antibodies specific for methylated, unmodified and acetylated histones . Representative results of quantified DNA derived from unstimulated (basal) and 5-aza-CdR- or TSA-stimulated DU145 and MCF-7 cells immunoprecipitated by antibodies specific for methylated histones (left diagrams) as well as for unmodified and acetylated histones (right diagrams). Examples of PTEN in DU145 cells (A, B), CD44 in MCF-7 cells (C, D), GLIPR1 in DU145 cells (E, F) and Cyclin D2 in DU145 cells (G, H) are shown. All values obtained were normalized and referred to 100% of the input DNA. IgG, negative control; H3K9, Lysine 9 of histone H3; H3K4, <t>Lysine</t> 4 of histone H3; H4K20, Lysine 20 of histone H4; H2A, histone H2A; H2B, histone H2B; me, mono-methylated; me2, dimethylated; me3, trimethylated; Ac, acetylated.
    Lysine 4, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    lysine 4 - by Bioz Stars, 2020-09
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    Dramatically decreased mono-ubiquitination of histone H2A lysine 119 (H2AK119u1) and phosphorylation of enhancer of zeste homolog 2 (Ezh2) methyltransferase in parthenogenetic blastocysts. Immunostaining was used to reveal that levels of total Ezh2 methyltransferase

    Journal: Stem Cells and Development

    Article Title: Epigenetic Disruptions of Histone Signatures for the Trophectoderm and Inner Cell Mass in Mouse Parthenogenetic Embryos

    doi: 10.1089/scd.2014.0310

    Figure Lengend Snippet: Dramatically decreased mono-ubiquitination of histone H2A lysine 119 (H2AK119u1) and phosphorylation of enhancer of zeste homolog 2 (Ezh2) methyltransferase in parthenogenetic blastocysts. Immunostaining was used to reveal that levels of total Ezh2 methyltransferase

    Article Snippet: After blocking, embryos were incubated at 4°C overnight with the following primary antibodies diluted in blocking solution: anti-acetylated histone H3 lysine 9 (rabbit polyclonal, 1:200 dilution; Abcam), anti-CARM1 (both mouse monoclonal and rabbit polyclonal, 1:200 dilution; Abcam), anti-Cdx2 (mouse monoclonal, 1:100 dilution; BioGenex), anti-dimethylated histone H3 arginine 26 (rabbit polyclonal, 1:200 dilution; Abcam), anti-Ezh2 (rabbit polyclonal, 1:200 dilution; Millipore), anti-mono-ubiquitinated histone H2A lysine 119 (mouse monoclonal, 1:200 dilution; Millipore), anti-phosphorylated Akt1 (phospho-S473) (rabbit polyclonal, 1:100 dilution; Abcam), anti-phosphorylated Ezh2 (phospho-S21) (rabbit polyclonal, 1:100 dilution; Abcam), anti-TCF7L2 (rabbit polyclonal, 1:100 dilution; Abcam), anti-trimethylated histone H3 lysine 4 (rabbit polyclonal, 1:200 dilution; Abcam), anti-trimethylated histone H3 lysine 9 (rabbit polyclonal, 1:200 dilution; Millipore), or anti-trimethylated histone H3 lysine 27 (rabbit polyclonal, 1:200 dilution; Millipore).

    Techniques: Immunostaining

    Comparison of global histone modification patterns of KSHV genomes in BrK.219 and PEL cells. Global patterns of histone H3 trimethylated at lysine 4 (H3K4-me3), lysine 9 (H3K9-me3), or lysine 27 (H3K27-me3) or acetylated at lysine 9 and/or lysine 14 (H3K27-me3

    Journal: Journal of Virology

    Article Title: Activation of the B Cell Antigen Receptor Triggers Reactivation of Latent Kaposi's Sarcoma-Associated Herpesvirus in B Cells

    doi: 10.1128/JVI.00506-13

    Figure Lengend Snippet: Comparison of global histone modification patterns of KSHV genomes in BrK.219 and PEL cells. Global patterns of histone H3 trimethylated at lysine 4 (H3K4-me3), lysine 9 (H3K9-me3), or lysine 27 (H3K27-me3) or acetylated at lysine 9 and/or lysine 14 (H3K27-me3

    Article Snippet: Briefly, chromatin was prepared from KSHV-infected BrK.219 cells and subjected to immunoprecipitation with antibodies specific for histone H3 trimethylated at lysine 4, lysine 9, or lysine 27 or acetylated at lysine 9 and/or lysine 14 (H3K4-me3, H3K9-me3, H3K27-me3, and H3K9/14-ac [Upstate/Millipore]) or with a matched isotype control antibody (IgG).

    Techniques: Modification

    Chromatin immunoprecipitation using antibodies specific for methylated, unmodified and acetylated histones . Representative results of quantified DNA derived from unstimulated (basal) and 5-aza-CdR- or TSA-stimulated DU145 and MCF-7 cells immunoprecipitated by antibodies specific for methylated histones (left diagrams) as well as for unmodified and acetylated histones (right diagrams). Examples of PTEN in DU145 cells (A, B), CD44 in MCF-7 cells (C, D), GLIPR1 in DU145 cells (E, F) and Cyclin D2 in DU145 cells (G, H) are shown. All values obtained were normalized and referred to 100% of the input DNA. IgG, negative control; H3K9, Lysine 9 of histone H3; H3K4, Lysine 4 of histone H3; H4K20, Lysine 20 of histone H4; H2A, histone H2A; H2B, histone H2B; me, mono-methylated; me2, dimethylated; me3, trimethylated; Ac, acetylated.

    Journal: BMC Cancer

    Article Title: Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by Methyl-CpG binding proteins and histone modifications

    doi: 10.1186/1471-2407-10-297

    Figure Lengend Snippet: Chromatin immunoprecipitation using antibodies specific for methylated, unmodified and acetylated histones . Representative results of quantified DNA derived from unstimulated (basal) and 5-aza-CdR- or TSA-stimulated DU145 and MCF-7 cells immunoprecipitated by antibodies specific for methylated histones (left diagrams) as well as for unmodified and acetylated histones (right diagrams). Examples of PTEN in DU145 cells (A, B), CD44 in MCF-7 cells (C, D), GLIPR1 in DU145 cells (E, F) and Cyclin D2 in DU145 cells (G, H) are shown. All values obtained were normalized and referred to 100% of the input DNA. IgG, negative control; H3K9, Lysine 9 of histone H3; H3K4, Lysine 4 of histone H3; H4K20, Lysine 20 of histone H4; H2A, histone H2A; H2B, histone H2B; me, mono-methylated; me2, dimethylated; me3, trimethylated; Ac, acetylated.

    Article Snippet: After blocking, the membrane was probed with a 1:500 or 1:1000 dilution of antibodies which are directed against the following human proteins: MBD1, MeCP2 (Abcam, Cambridge, UK), MBD2, monomethylated lysine 9 of histone H3 (H3K9me), dimethylated lysine 9 of histone H3 (H3K9me2), trimethylated lysine 9 of histone H3 (H3K9me3), monomethylated lysine 4 of histone H3 (H3K4me), dimethylated lysine 4 of histone H3 (H3K4me2), trimethylated lysine 4 of histone H3 (H3K4me3), monomethylated lysine 20 of histone H4 (H4K20me), dimethylated lysine 20 of histone H4 (H4K20me2), trimethylated lysine 20 of histone H4 (H4K20me3) (Millipore, Schwalbach, Germany).

    Techniques: Chromatin Immunoprecipitation, Methylation, Derivative Assay, Immunoprecipitation, Negative Control