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Millipore hescs
Gene and protein expression analysis of definitive endoderm (DE) markers in experimental and control groups at day 4. A. Diagrammatic representation of the experimental groups (Spd-A50/A25 and Spd-IDE1/2), positive control (W/A100-A100) and negative control (DMSO) for endoderm induction of human embryonic stem cells <t>(hESCs).</t> B. Lineage-specific gene expression analysis of Spd-A50-treated <t>Royan</t> H6 human embryonic stem cells (hESC-RH6). In control groups, hESCs were treated with 0.1% dimethyl sulfoxide (DMSO) for 4 days and considered as the negative control. Cells were treated with Wnt3a and activin A for the first day, followed by treatment with activin A for the next three days (W/A100-A100) as the positive control. As determined by Q-PCR, the DE markers SOX17, FOXA2 and CXCR4 highly expressed in Spd-A50-treated Royan H6 hESCs while SOX7 [visceral endoderm (VE) marker], SOX1 (neuroectoderm marker) and OCT4 (pluripotency marker) had low levels of expression. The target gene expression level was normalized to GAPDH and presented relative to hESC. Data are presented as mean ± SD. C. Immunofluorescent staining of Spd-A50-treated human embryonic stem cells (hESCs) showed SOX17+ (green) populations comparable to that of W/A100-A100- (positive control) cells. DAPI; 4=,6-diamidino-2-phenylindole.
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1) Product Images from "Stauprimide Priming of Human Embryonic Stem Cells toward Definitive Endoderm"

Article Title: Stauprimide Priming of Human Embryonic Stem Cells toward Definitive Endoderm

Journal: Cell Journal (Yakhteh)

doi:

Gene and protein expression analysis of definitive endoderm (DE) markers in experimental and control groups at day 4. A. Diagrammatic representation of the experimental groups (Spd-A50/A25 and Spd-IDE1/2), positive control (W/A100-A100) and negative control (DMSO) for endoderm induction of human embryonic stem cells (hESCs). B. Lineage-specific gene expression analysis of Spd-A50-treated Royan H6 human embryonic stem cells (hESC-RH6). In control groups, hESCs were treated with 0.1% dimethyl sulfoxide (DMSO) for 4 days and considered as the negative control. Cells were treated with Wnt3a and activin A for the first day, followed by treatment with activin A for the next three days (W/A100-A100) as the positive control. As determined by Q-PCR, the DE markers SOX17, FOXA2 and CXCR4 highly expressed in Spd-A50-treated Royan H6 hESCs while SOX7 [visceral endoderm (VE) marker], SOX1 (neuroectoderm marker) and OCT4 (pluripotency marker) had low levels of expression. The target gene expression level was normalized to GAPDH and presented relative to hESC. Data are presented as mean ± SD. C. Immunofluorescent staining of Spd-A50-treated human embryonic stem cells (hESCs) showed SOX17+ (green) populations comparable to that of W/A100-A100- (positive control) cells. DAPI; 4=,6-diamidino-2-phenylindole.
Figure Legend Snippet: Gene and protein expression analysis of definitive endoderm (DE) markers in experimental and control groups at day 4. A. Diagrammatic representation of the experimental groups (Spd-A50/A25 and Spd-IDE1/2), positive control (W/A100-A100) and negative control (DMSO) for endoderm induction of human embryonic stem cells (hESCs). B. Lineage-specific gene expression analysis of Spd-A50-treated Royan H6 human embryonic stem cells (hESC-RH6). In control groups, hESCs were treated with 0.1% dimethyl sulfoxide (DMSO) for 4 days and considered as the negative control. Cells were treated with Wnt3a and activin A for the first day, followed by treatment with activin A for the next three days (W/A100-A100) as the positive control. As determined by Q-PCR, the DE markers SOX17, FOXA2 and CXCR4 highly expressed in Spd-A50-treated Royan H6 hESCs while SOX7 [visceral endoderm (VE) marker], SOX1 (neuroectoderm marker) and OCT4 (pluripotency marker) had low levels of expression. The target gene expression level was normalized to GAPDH and presented relative to hESC. Data are presented as mean ± SD. C. Immunofluorescent staining of Spd-A50-treated human embryonic stem cells (hESCs) showed SOX17+ (green) populations comparable to that of W/A100-A100- (positive control) cells. DAPI; 4=,6-diamidino-2-phenylindole.

Techniques Used: Expressing, Positive Control, Negative Control, Polymerase Chain Reaction, Marker, Staining

2) Product Images from "Ubp8 and SAGA Regulate Snf1 AMP Kinase Activity ▿"

Article Title: Ubp8 and SAGA Regulate Snf1 AMP Kinase Activity ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01350-10

Snf1 interacts with Ubp8 in vivo . (A and B) Whole-cell extracts were collected from yeast strains expressing an empty vector (lanes 1 and 5), Ubp8 with an N-terminal myc tag (lanes 2 and 6), or Snf1 with a C-terminal FLAG tag (lanes 3 and 7) or coexpressing
Figure Legend Snippet: Snf1 interacts with Ubp8 in vivo . (A and B) Whole-cell extracts were collected from yeast strains expressing an empty vector (lanes 1 and 5), Ubp8 with an N-terminal myc tag (lanes 2 and 6), or Snf1 with a C-terminal FLAG tag (lanes 3 and 7) or coexpressing

Techniques Used: In Vivo, Expressing, Plasmid Preparation, FLAG-tag

SAGA deubiquitinates Snf1 in vitro . (A) Ubiquitinated histone H2B was purified in strains expressing a FLAG epitope-tagged H2B and a 3×HA epitope-tagged ubiquitin. Ubiquitination of purified H2B was confirmed by Western blotting with anti-H2B,
Figure Legend Snippet: SAGA deubiquitinates Snf1 in vitro . (A) Ubiquitinated histone H2B was purified in strains expressing a FLAG epitope-tagged H2B and a 3×HA epitope-tagged ubiquitin. Ubiquitination of purified H2B was confirmed by Western blotting with anti-H2B,

Techniques Used: In Vitro, Purification, Expressing, FLAG-tag, Western Blot

3) Product Images from "Tumour-specific cytotoxic T lymphocyte activity in Th2-type S?zary syndrome: its enhancement by interferon-gamma (IFN-?) and IL-12 and fluctuations in association with disease activity"

Article Title: Tumour-specific cytotoxic T lymphocyte activity in Th2-type S?zary syndrome: its enhancement by interferon-gamma (IFN-?) and IL-12 and fluctuations in association with disease activity

Journal: Clinical and Experimental Immunology

doi: 10.1046/j.1365-2249.1998.00599.x

Enhancement of cytotoxicity of CD8 + cells by rIL-12 and/or rIL-2. CD8 + cells of the Sézary syndrome (SzS) patient at time point C were cultured in the presence of rIL-2 (50 U/ml), rIL-12 (1 or 10 ng/ml), rIL-12 (1 or 10 ng/ml) combined with rIL-2 (50 U/ml), rIFN-γ (1000 or 5000 U/ml), and rIFN-γ (1000 or 5000 U/ml) combined with rIL-2 (50 U/ml) with mitomycin-C- treated purified Sézary cells. Cytotoxicity of the cultured CD8 + cells (effector cells) was assayed by 51 Cr-release of labelled Sézary cell (target cells) at an effector/target ratio of 10. Data are expressed as mean ± s.e.m. of the results of duplicate experiments.
Figure Legend Snippet: Enhancement of cytotoxicity of CD8 + cells by rIL-12 and/or rIL-2. CD8 + cells of the Sézary syndrome (SzS) patient at time point C were cultured in the presence of rIL-2 (50 U/ml), rIL-12 (1 or 10 ng/ml), rIL-12 (1 or 10 ng/ml) combined with rIL-2 (50 U/ml), rIFN-γ (1000 or 5000 U/ml), and rIFN-γ (1000 or 5000 U/ml) combined with rIL-2 (50 U/ml) with mitomycin-C- treated purified Sézary cells. Cytotoxicity of the cultured CD8 + cells (effector cells) was assayed by 51 Cr-release of labelled Sézary cell (target cells) at an effector/target ratio of 10. Data are expressed as mean ± s.e.m. of the results of duplicate experiments.

Techniques Used: Cell Culture, Purification

Relationships of cytotoxic activity of cultured CD8 + cells, circulating CD8 + cell percentage, and serum lactate dehydrogenase (LDH) level in the patient's clinical course. (a) CD8 + cells were obtained from the patient at four different time points (A–D) at intervals of 10–14 days during a 2-month clinical period. CD8 + cells separated at each time point were cultured in the presence of rIL-2 (50 U/ml) (□), or rIL-2 (50 U/ml) combined with rIFN-γ (500 U/ml (▪), 1000 U/ml (○), or 5000 U/ml (•)) with mitomycin-C-treated purified Sézary cells. Cytotoxicity of the cultured CD8 + cells (effector cells) was assayed by 51 Cr-release of labelled Sézary tumour cells (target cells) at the indicated effector/target ratios. (b) CD8 + cells separated at each time point were cultured in medium supplemented with rIL-2 (50 U/ml) and rIFN-γ (5000 U/ml). Sézary cell-specific cytotoxicity was measured by cytolytic assay at an effector/target ratio of 10 (•). PBMC were stained by fluorescein-conjugated anti-CD8 MoAb and subjected to flow cytometric analysis (○). PBMC were double-stained by fluorescein-conjugated anti-CD7 MoAb and PE-conjugated anti-CD4 MoAb, and percentage of CD4 + CD7 − cells was calculated by flow cytometry (□). LDH levels in sera were measured at each time point (▪).
Figure Legend Snippet: Relationships of cytotoxic activity of cultured CD8 + cells, circulating CD8 + cell percentage, and serum lactate dehydrogenase (LDH) level in the patient's clinical course. (a) CD8 + cells were obtained from the patient at four different time points (A–D) at intervals of 10–14 days during a 2-month clinical period. CD8 + cells separated at each time point were cultured in the presence of rIL-2 (50 U/ml) (□), or rIL-2 (50 U/ml) combined with rIFN-γ (500 U/ml (▪), 1000 U/ml (○), or 5000 U/ml (•)) with mitomycin-C-treated purified Sézary cells. Cytotoxicity of the cultured CD8 + cells (effector cells) was assayed by 51 Cr-release of labelled Sézary tumour cells (target cells) at the indicated effector/target ratios. (b) CD8 + cells separated at each time point were cultured in medium supplemented with rIL-2 (50 U/ml) and rIFN-γ (5000 U/ml). Sézary cell-specific cytotoxicity was measured by cytolytic assay at an effector/target ratio of 10 (•). PBMC were stained by fluorescein-conjugated anti-CD8 MoAb and subjected to flow cytometric analysis (○). PBMC were double-stained by fluorescein-conjugated anti-CD7 MoAb and PE-conjugated anti-CD4 MoAb, and percentage of CD4 + CD7 − cells was calculated by flow cytometry (□). LDH levels in sera were measured at each time point (▪).

Techniques Used: Activity Assay, Cell Culture, Purification, Staining, Flow Cytometry, Cytometry

4) Product Images from "Mesenchymal stem cells derived from human iPS cells via mesoderm and neuroepithelium have different features and therapeutic potentials"

Article Title: Mesenchymal stem cells derived from human iPS cells via mesoderm and neuroepithelium have different features and therapeutic potentials

Journal: PLoS ONE

doi: 10.1371/journal.pone.0200790

DNA microarray analysis of PSP-MSCs and RA-Pα-MSCs. (A-C): Hierarchical clustering of gene sets signatures, pluripotent markers (A), MSC markers (B) and paracrine factors (C), in various MSCs and N1-12 iPSCs. The datasets of all genes investigated were clustered according to Euclidean distance metrics. The labels represent the following cells: N1-12; N1-12 iPS cells, BM-MSC; BM-MSC (PRC-010) purchased from Bay bioscience Co.; N1-12 PSP; PSP-MSC derived from N1-12, 201B7 PSP; PSP-MSC derived from 201B7, N1-12 RA-Pα; RA-Pα-MSC derived from N1-12, 201B7 RA-Pα; RA-Pa-MSC derived from 201B7. (D): Principal component analysis. All datasets were classified into three principal components, PC2 (25.43%), PC3 (15.34%), and PC4 (7.81%), and were simplified into three-dimensional scores. (E, F): Gene ontology (GO) analysis of 286 commonly upregulated data sets for PSP-MSC (E) and 359 data sets for RA-Pα-MSC (F). The top-ten GO terms are listed. GO terms were detected with a cutoff P-value of 0.1. Values are–log10 corrected P-value. Red color indicates different GO terms between (E) and (F).
Figure Legend Snippet: DNA microarray analysis of PSP-MSCs and RA-Pα-MSCs. (A-C): Hierarchical clustering of gene sets signatures, pluripotent markers (A), MSC markers (B) and paracrine factors (C), in various MSCs and N1-12 iPSCs. The datasets of all genes investigated were clustered according to Euclidean distance metrics. The labels represent the following cells: N1-12; N1-12 iPS cells, BM-MSC; BM-MSC (PRC-010) purchased from Bay bioscience Co.; N1-12 PSP; PSP-MSC derived from N1-12, 201B7 PSP; PSP-MSC derived from 201B7, N1-12 RA-Pα; RA-Pα-MSC derived from N1-12, 201B7 RA-Pα; RA-Pa-MSC derived from 201B7. (D): Principal component analysis. All datasets were classified into three principal components, PC2 (25.43%), PC3 (15.34%), and PC4 (7.81%), and were simplified into three-dimensional scores. (E, F): Gene ontology (GO) analysis of 286 commonly upregulated data sets for PSP-MSC (E) and 359 data sets for RA-Pα-MSC (F). The top-ten GO terms are listed. GO terms were detected with a cutoff P-value of 0.1. Values are–log10 corrected P-value. Red color indicates different GO terms between (E) and (F).

Techniques Used: Microarray, Derivative Assay

5) Product Images from "Mesenchymal stem cells derived from human iPS cells via mesoderm and neuroepithelium have different features and therapeutic potentials"

Article Title: Mesenchymal stem cells derived from human iPS cells via mesoderm and neuroepithelium have different features and therapeutic potentials

Journal: PLoS ONE

doi: 10.1371/journal.pone.0200790

DNA microarray analysis of PSP-MSCs and RA-Pα-MSCs. (A-C): Hierarchical clustering of gene sets signatures, pluripotent markers (A), MSC markers (B) and paracrine factors (C), in various MSCs and N1-12 iPSCs. The datasets of all genes investigated were clustered according to Euclidean distance metrics. The labels represent the following cells: N1-12; N1-12 iPS cells, BM-MSC; BM-MSC (PRC-010) purchased from Bay bioscience Co.; N1-12 PSP; PSP-MSC derived from N1-12, 201B7 PSP; PSP-MSC derived from 201B7, N1-12 RA-Pα; RA-Pα-MSC derived from N1-12, 201B7 RA-Pα; RA-Pa-MSC derived from 201B7. (D): Principal component analysis. All datasets were classified into three principal components, PC2 (25.43%), PC3 (15.34%), and PC4 (7.81%), and were simplified into three-dimensional scores. (E, F): Gene ontology (GO) analysis of 286 commonly upregulated data sets for PSP-MSC (E) and 359 data sets for RA-Pα-MSC (F). The top-ten GO terms are listed. GO terms were detected with a cutoff P-value of 0.1. Values are–log10 corrected P-value. Red color indicates different GO terms between (E) and (F).
Figure Legend Snippet: DNA microarray analysis of PSP-MSCs and RA-Pα-MSCs. (A-C): Hierarchical clustering of gene sets signatures, pluripotent markers (A), MSC markers (B) and paracrine factors (C), in various MSCs and N1-12 iPSCs. The datasets of all genes investigated were clustered according to Euclidean distance metrics. The labels represent the following cells: N1-12; N1-12 iPS cells, BM-MSC; BM-MSC (PRC-010) purchased from Bay bioscience Co.; N1-12 PSP; PSP-MSC derived from N1-12, 201B7 PSP; PSP-MSC derived from 201B7, N1-12 RA-Pα; RA-Pα-MSC derived from N1-12, 201B7 RA-Pα; RA-Pa-MSC derived from 201B7. (D): Principal component analysis. All datasets were classified into three principal components, PC2 (25.43%), PC3 (15.34%), and PC4 (7.81%), and were simplified into three-dimensional scores. (E, F): Gene ontology (GO) analysis of 286 commonly upregulated data sets for PSP-MSC (E) and 359 data sets for RA-Pα-MSC (F). The top-ten GO terms are listed. GO terms were detected with a cutoff P-value of 0.1. Values are–log10 corrected P-value. Red color indicates different GO terms between (E) and (F).

Techniques Used: Microarray, Derivative Assay

Induction of hiPSC-derived MSCs under mesodermal and neuroepithelial differentiation conditions. (A, B): The proportions of PDGFRα and VEGFR2 expression in differentiated N1-12 cells on day 2, 4, 6 and 8 under the mesodermal differentiation condition. Representative data (A) and graph (B). Number indicates the percentage of each population (A). Experiments were conducted three times (mean ± SD). (C): Quantitative PCR (qPCR) analysis of the relative mRNA levels of NANOG and OCT3/4 as multipotent markers, VIMENTIN and PDGFRβ for mesenchyme, BRACHYURY for mesoderm, and SOX1 for neuroepithelium during the mesodermal (left) and neuroepithelial (right) differentiations. Symbols: d-number indicates day-number of differentiation, PSP; immediately after sorting on day 6 under the mesoderm differentiation, RA-Pα: immediately after sorting on day 10 under the neuroepithelial differentiation, P3 or P6; passage 3 or 6 after sorting. Each value represents the mean fold compared to day-0 undifferentiated iPSCs. Each experiment was conducted three times (mean ± SD). (D): Bright-light image of PSP-MSC and RA-Pα cells on day 21 after sorting. Scale bar: 200 μm. (E): The proportion of PDGFRα and VEGFR2 expressions in day-10 differentiated N1-12 under the neuroepithelial differentiation condition; number indicates the percentage of each population.
Figure Legend Snippet: Induction of hiPSC-derived MSCs under mesodermal and neuroepithelial differentiation conditions. (A, B): The proportions of PDGFRα and VEGFR2 expression in differentiated N1-12 cells on day 2, 4, 6 and 8 under the mesodermal differentiation condition. Representative data (A) and graph (B). Number indicates the percentage of each population (A). Experiments were conducted three times (mean ± SD). (C): Quantitative PCR (qPCR) analysis of the relative mRNA levels of NANOG and OCT3/4 as multipotent markers, VIMENTIN and PDGFRβ for mesenchyme, BRACHYURY for mesoderm, and SOX1 for neuroepithelium during the mesodermal (left) and neuroepithelial (right) differentiations. Symbols: d-number indicates day-number of differentiation, PSP; immediately after sorting on day 6 under the mesoderm differentiation, RA-Pα: immediately after sorting on day 10 under the neuroepithelial differentiation, P3 or P6; passage 3 or 6 after sorting. Each value represents the mean fold compared to day-0 undifferentiated iPSCs. Each experiment was conducted three times (mean ± SD). (D): Bright-light image of PSP-MSC and RA-Pα cells on day 21 after sorting. Scale bar: 200 μm. (E): The proportion of PDGFRα and VEGFR2 expressions in day-10 differentiated N1-12 under the neuroepithelial differentiation condition; number indicates the percentage of each population.

Techniques Used: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction

6) Product Images from "A small-molecule inhibitor suppresses the tumor-associated mitochondrial NAD(P)+-dependent malic enzyme (ME2) and induces cellular senescence"

Article Title: A small-molecule inhibitor suppresses the tumor-associated mitochondrial NAD(P)+-dependent malic enzyme (ME2) and induces cellular senescence

Journal: Oncotarget

doi:

Chemical structure of embonic acid (EA) and inhibitory effect of EA on human m-NAD(P)-ME and c-NADP-ME A. Chemical structure of EA. B. The assay mixture contained 40 mM malate, 10 mM MgCl 2 , 2 mM NAD + or NADP + and 50 mM Tris-HCl (pH 7.5) with various concentrations of EA. The concentrations of EA ranged from 0 to 75 μM. Closed circles, m-NAD(P)-ME; open circles, c-NADP-ME.
Figure Legend Snippet: Chemical structure of embonic acid (EA) and inhibitory effect of EA on human m-NAD(P)-ME and c-NADP-ME A. Chemical structure of EA. B. The assay mixture contained 40 mM malate, 10 mM MgCl 2 , 2 mM NAD + or NADP + and 50 mM Tris-HCl (pH 7.5) with various concentrations of EA. The concentrations of EA ranged from 0 to 75 μM. Closed circles, m-NAD(P)-ME; open circles, c-NADP-ME.

Techniques Used:

7) Product Images from "MyD88- and TRIF-Independent Induction of Type I Interferon Drives Naive B Cell Accumulation but Not Loss of Lymph Node Architecture in Lyme Disease"

Article Title: MyD88- and TRIF-Independent Induction of Type I Interferon Drives Naive B Cell Accumulation but Not Loss of Lymph Node Architecture in Lyme Disease

Journal: Infection and Immunity

doi: 10.1128/IAI.00969-13

Lymph node-homing chemokine expression following infection with host-adapted B. burgdorferi . Shown are mean relative expression levels + SD of the indicated chemokines measured by qRT-PCR in lymph nodes of mice infected for 10 or 22 days compared to lymph nodes from uninfected mice ( n = 4/group). Data are normalized for RNA input using the GAPDH housekeeping gene.
Figure Legend Snippet: Lymph node-homing chemokine expression following infection with host-adapted B. burgdorferi . Shown are mean relative expression levels + SD of the indicated chemokines measured by qRT-PCR in lymph nodes of mice infected for 10 or 22 days compared to lymph nodes from uninfected mice ( n = 4/group). Data are normalized for RNA input using the GAPDH housekeeping gene.

Techniques Used: Expressing, Infection, Quantitative RT-PCR, Mouse Assay

Gene expression changes in lymph nodes after infection and immunization with B. burgdorferi . Shown are mean relative expression levels + SD of cytokines measured by qRT-PCR of lymph nodes of mice infected or immunized for 4 days compared to lymph nodes from uninfected mice ( n = 4/group). Data are normalized for RNA input using the GAPDH housekeeping gene. Statistical analysis used one-way ANOVA with the Dunnett posttest: *, P
Figure Legend Snippet: Gene expression changes in lymph nodes after infection and immunization with B. burgdorferi . Shown are mean relative expression levels + SD of cytokines measured by qRT-PCR of lymph nodes of mice infected or immunized for 4 days compared to lymph nodes from uninfected mice ( n = 4/group). Data are normalized for RNA input using the GAPDH housekeeping gene. Statistical analysis used one-way ANOVA with the Dunnett posttest: *, P

Techniques Used: Expressing, Infection, Quantitative RT-PCR, Mouse Assay

B. burgdorferi infection does not impair chemokine-directed B cell migration. (A to D) Representative 5% contour plots with outliers, indicating mean frequencies of cell populations stained as indicated following gating for live, single-lymphocyte-size cells from lymph nodes of noninfected and day 10 B. burgdorferi -infected mice ( n = 4). Small graphs are for fluorescence-minus-one (FMO) control stains. (A and B) Line graphs indicate mean frequencies + SD ( n = 4/time point) of CXCR4 + cells at the indicated time points before and after infection (A) and means + SD ( n = 4/time point) of median fluorescence intensities (MFIs) for CXCR5 + staining on CD19 + B cells at the indicated times after infection with B. burgdorferi (B). (C and D) Mean frequencies ± SD of CXCR4 + CD138 − non-germinal center B cells and CXCR4 + CD138 + plasma cells as percentages of CD19 + B cells (C) and of germinal center B cells, identified as CD24 hi CD38 lo CD19 + CD45R + (D, left), and CXCR4 and CXCR5 expression by germinal center B cells ( n = 6) (D, right). (E and F) Mean frequencies + SD from quadruplicate cultures of migrated CD19 + cells compared to total input cells following ex vivo transwell migration experiments with chemokine ligand CXCL12 (E) or CXCL13 (F, left) and frequencies + SD of migrated B cells from day 10-infected, day 10-immunized, and control lymph nodes (F, right). Statistical analysis used one-way ANOVA with the Dunnett posttest: *, P
Figure Legend Snippet: B. burgdorferi infection does not impair chemokine-directed B cell migration. (A to D) Representative 5% contour plots with outliers, indicating mean frequencies of cell populations stained as indicated following gating for live, single-lymphocyte-size cells from lymph nodes of noninfected and day 10 B. burgdorferi -infected mice ( n = 4). Small graphs are for fluorescence-minus-one (FMO) control stains. (A and B) Line graphs indicate mean frequencies + SD ( n = 4/time point) of CXCR4 + cells at the indicated time points before and after infection (A) and means + SD ( n = 4/time point) of median fluorescence intensities (MFIs) for CXCR5 + staining on CD19 + B cells at the indicated times after infection with B. burgdorferi (B). (C and D) Mean frequencies ± SD of CXCR4 + CD138 − non-germinal center B cells and CXCR4 + CD138 + plasma cells as percentages of CD19 + B cells (C) and of germinal center B cells, identified as CD24 hi CD38 lo CD19 + CD45R + (D, left), and CXCR4 and CXCR5 expression by germinal center B cells ( n = 6) (D, right). (E and F) Mean frequencies + SD from quadruplicate cultures of migrated CD19 + cells compared to total input cells following ex vivo transwell migration experiments with chemokine ligand CXCL12 (E) or CXCL13 (F, left) and frequencies + SD of migrated B cells from day 10-infected, day 10-immunized, and control lymph nodes (F, right). Statistical analysis used one-way ANOVA with the Dunnett posttest: *, P

Techniques Used: Infection, Migration, Staining, Mouse Assay, Fluorescence, Expressing, Ex Vivo

Alteration of tissue architecture and B cell accumulation in the lymph node following infection with host-adapted B. burgdorferi . (A) H E staining of lymph nodes on days 3 to 8 postinfection. Black arrows indicate B cell follicles (original magnification, ×10), representative of n = 2/group. (B) H E staining of lymph nodes from mice taken at day 10 immunization (left) or infection (right) with B. burgdorferi . White arrows indicate germinal centers with B cell follicles, and black arrows indicate primary B cell follicles without germinal centers (original magnification, ×10), representative of n = 2/group. (C) Shown are representative 5% contour plots and outliers generated by flow cytometry on draining inguinal lymph nodes collected at the indicated times after infection and stained to determine the frequency of CD3 + CD4 + T cells, CD19 + B cells, CD4 − CD19 − cells (live, single cells and CD19 − CD3 − CD8 − ). Numbers represent mean ± SD of the gated cells ( n = 4). Data are representative of two independent experiments.
Figure Legend Snippet: Alteration of tissue architecture and B cell accumulation in the lymph node following infection with host-adapted B. burgdorferi . (A) H E staining of lymph nodes on days 3 to 8 postinfection. Black arrows indicate B cell follicles (original magnification, ×10), representative of n = 2/group. (B) H E staining of lymph nodes from mice taken at day 10 immunization (left) or infection (right) with B. burgdorferi . White arrows indicate germinal centers with B cell follicles, and black arrows indicate primary B cell follicles without germinal centers (original magnification, ×10), representative of n = 2/group. (C) Shown are representative 5% contour plots and outliers generated by flow cytometry on draining inguinal lymph nodes collected at the indicated times after infection and stained to determine the frequency of CD3 + CD4 + T cells, CD19 + B cells, CD4 − CD19 − cells (live, single cells and CD19 − CD3 − CD8 − ). Numbers represent mean ± SD of the gated cells ( n = 4). Data are representative of two independent experiments.

Techniques Used: Infection, Staining, Mouse Assay, Generated, Flow Cytometry, Cytometry

B cell accumulation is type I IFN dependent but MyD88 and TRIF independent. (A) Mean frequencies + SD of CD19 + B cells, CD4 + T cells, and CD8 + T cells as assessed by flow cytometry in wild-type (WT) and MyD88 −/− mice ( n = 8/group) at day 10 postinfection. n.s., not significant. (B) Mean frequencies and SD of CD19 + B cells, CD4 + T cells, and CD8 + T cells as assessed by flow cytometry in WT and MyD88 −/− × TRIF −/− mice ( n = 4/group) on day 15 postinfection. (C) H E staining of representative WT, IFNAR −/− , and MyD88 −/− mice ( n = 2/group) at day 10 postinfection (magnification, ×10). (D) Mean relative expression levels (+ SEM) compared to GAPDH of type I IFN-induced genes (IFIT1 and IFIT2), IFN-α4, and IFN-β measured by qRT-PCR of lymph nodes from day 0 (uninfected, black bars), day 10 and 15 B. burgdorferi -infected (white bars), and sham-infected (gray bars) WT mice ( n = 4/group). (E) Frequencies + SD of plasmacytoid DC in WT mice ( n = 8/group) at day 10 after infection or immunization with B. burgdorferi lysate in adjuvant. (F) Mean frequencies + SD ( n = 4 to 8/group) of CD19 + B cells in draining lymph nodes of WT (dashed line) and IFNAR −/− (solid line) mice at the indicated times after infection.
Figure Legend Snippet: B cell accumulation is type I IFN dependent but MyD88 and TRIF independent. (A) Mean frequencies + SD of CD19 + B cells, CD4 + T cells, and CD8 + T cells as assessed by flow cytometry in wild-type (WT) and MyD88 −/− mice ( n = 8/group) at day 10 postinfection. n.s., not significant. (B) Mean frequencies and SD of CD19 + B cells, CD4 + T cells, and CD8 + T cells as assessed by flow cytometry in WT and MyD88 −/− × TRIF −/− mice ( n = 4/group) on day 15 postinfection. (C) H E staining of representative WT, IFNAR −/− , and MyD88 −/− mice ( n = 2/group) at day 10 postinfection (magnification, ×10). (D) Mean relative expression levels (+ SEM) compared to GAPDH of type I IFN-induced genes (IFIT1 and IFIT2), IFN-α4, and IFN-β measured by qRT-PCR of lymph nodes from day 0 (uninfected, black bars), day 10 and 15 B. burgdorferi -infected (white bars), and sham-infected (gray bars) WT mice ( n = 4/group). (E) Frequencies + SD of plasmacytoid DC in WT mice ( n = 8/group) at day 10 after infection or immunization with B. burgdorferi lysate in adjuvant. (F) Mean frequencies + SD ( n = 4 to 8/group) of CD19 + B cells in draining lymph nodes of WT (dashed line) and IFNAR −/− (solid line) mice at the indicated times after infection.

Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, Staining, Expressing, Quantitative RT-PCR, Infection

8) Product Images from "Importin 7 and Importin ?/Importin ? Are Nuclear Import Receptors for the Glucocorticoid Receptor"

Article Title: Importin 7 and Importin ?/Importin ? Are Nuclear Import Receptors for the Glucocorticoid Receptor

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E03-11-0839

Reconstitution of GR nuclear import in vitro. Bar, 10 μM. (A) GFP-GST-407-525 (0.5 μM) was incubated with permeabilized cells, 2 mg/ml HeLa extract, ATP mix, 6 μM RanQ69L, or apyrase for 30 min at 30°C or 4°C, as
Figure Legend Snippet: Reconstitution of GR nuclear import in vitro. Bar, 10 μM. (A) GFP-GST-407-525 (0.5 μM) was incubated with permeabilized cells, 2 mg/ml HeLa extract, ATP mix, 6 μM RanQ69L, or apyrase for 30 min at 30°C or 4°C, as

Techniques Used: In Vitro, Incubation

9) Product Images from "Therapeutic effect of the potent IL-12/IL-23 inhibitor STA-5326 on experimental autoimmune uveoretinitis"

Article Title: Therapeutic effect of the potent IL-12/IL-23 inhibitor STA-5326 on experimental autoimmune uveoretinitis

Journal: Arthritis Research & Therapy

doi: 10.1186/ar2530

Antigen specific proliferation is decreased in lymph node cells of STA-5326-treated mice, and IL-17 production and the proportion of Th17 cells from draining lymph nodes are significantly reduced in STA-5326-treated mice . (a) Antigen specific proliferation of draining lymph nodes in STA-5326-treated or vehicle-treated mice. Immunized mice were treated with 5 mg/kg or 20 mg/kg STA-5326 or vehicle from day 0 to day 14 after immunization. Draining lymph node cells collected on day 18 after immunization were pooled within each group. Cultures were stimulated with 10 μg/ml human experimental autoimmune uveoretinitis (IRBP) peptide 1–20 for 72 hours and pulsed with bromodeoxyuridine for the last 24 hours. (b-d) Cytokine production of interferon (IFN) γ, interleukin (IL) 17 and IL-4 by draining lymph node cells from STA-5326-treated or vehicle-treated mice. Immunized mice were treated with 5 mg/kg or 20 mg/kg STA-5326 or vehicle from day 0 to day 14 after immunization. Draining lymph node cells collected on day 18 after immunization were pooled within each group. Cultures were stimulated with 10 μg/ml IRBP 1–20 for 72 hours, and supernatants collected at 72 hours were assayed by ELISA. (a-d) Statistical analysis was performed using Student;s t -test. (e and f) Intracellular cytokine staining of draining lymph node cells in 20 mg/kg STA-5326 or vehicle-treated mice. Draining lymph node cells collected on day 18 were stimulated with IRBP 1–20 for 72 hours, and the cultured cells were incubated with PMA plus ionomycin and brefeldin A and stained with CD4, CD8 and intracellular IFN-γ and IL-17. The percentage shown in the upper right quadrant is for IFN-γ or IL-17 positive cells in CD4 + T cells.
Figure Legend Snippet: Antigen specific proliferation is decreased in lymph node cells of STA-5326-treated mice, and IL-17 production and the proportion of Th17 cells from draining lymph nodes are significantly reduced in STA-5326-treated mice . (a) Antigen specific proliferation of draining lymph nodes in STA-5326-treated or vehicle-treated mice. Immunized mice were treated with 5 mg/kg or 20 mg/kg STA-5326 or vehicle from day 0 to day 14 after immunization. Draining lymph node cells collected on day 18 after immunization were pooled within each group. Cultures were stimulated with 10 μg/ml human experimental autoimmune uveoretinitis (IRBP) peptide 1–20 for 72 hours and pulsed with bromodeoxyuridine for the last 24 hours. (b-d) Cytokine production of interferon (IFN) γ, interleukin (IL) 17 and IL-4 by draining lymph node cells from STA-5326-treated or vehicle-treated mice. Immunized mice were treated with 5 mg/kg or 20 mg/kg STA-5326 or vehicle from day 0 to day 14 after immunization. Draining lymph node cells collected on day 18 after immunization were pooled within each group. Cultures were stimulated with 10 μg/ml IRBP 1–20 for 72 hours, and supernatants collected at 72 hours were assayed by ELISA. (a-d) Statistical analysis was performed using Student;s t -test. (e and f) Intracellular cytokine staining of draining lymph node cells in 20 mg/kg STA-5326 or vehicle-treated mice. Draining lymph node cells collected on day 18 were stimulated with IRBP 1–20 for 72 hours, and the cultured cells were incubated with PMA plus ionomycin and brefeldin A and stained with CD4, CD8 and intracellular IFN-γ and IL-17. The percentage shown in the upper right quadrant is for IFN-γ or IL-17 positive cells in CD4 + T cells.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Staining, Cell Culture, Incubation

10) Product Images from "Identification of the Immunodominant Epitope Region in Phospholipase A2 Receptor-Mediating Autoantibody Binding in Idiopathic Membranous Nephropathy"

Article Title: Identification of the Immunodominant Epitope Region in Phospholipase A2 Receptor-Mediating Autoantibody Binding in Idiopathic Membranous Nephropathy

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2013121315

Comparison of the PLA2 R 1–3 Construct and the Full-Length PLA2 R for Patient Sera Immunoscreening
Figure Legend Snippet: Comparison of the PLA2 R 1–3 Construct and the Full-Length PLA2 R for Patient Sera Immunoscreening

Techniques Used: Construct

11) Product Images from "Cholesterol Oxidase Is Indispensable in the Pathogenesis of Mycobacterium tuberculosis"

Article Title: Cholesterol Oxidase Is Indispensable in the Pathogenesis of Mycobacterium tuberculosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0073333

Percentage of macrophages that ingest Mtb. (A) Macrophages were infected with FITC-labeled wild-type or Δ choD strains for 2 hours. (B and C) Macrophages were treated with IRAK1/4 inhibitor, anti-TLR2 mAb, or anti-CR3 mAb for 1 hour, and then were infected with (B) opsonized or (C) non-opsonized FITC-labeled wild-type or Δ choD strains for 2 hours. In all samples, macrophages with ingested bacteria were counted using a fluorescence microscope. All data are presented as the percentage of macrophages involved in phagocytosis, expressed as means ± SEMs (*p≤0.02, Mtb strain vs. Mtb strain+IRAK1/4 inhibitor or anti-TLR2 mAb or anti-CR3 mAb, # p≤0.03, Δ choD +anti-CR3 mAb vs. Δ choD +anti-TLR2 mAb,; Mann-Whitney U test). Data are presented from five independent experiments. Each experiment was carried out in duplicate.
Figure Legend Snippet: Percentage of macrophages that ingest Mtb. (A) Macrophages were infected with FITC-labeled wild-type or Δ choD strains for 2 hours. (B and C) Macrophages were treated with IRAK1/4 inhibitor, anti-TLR2 mAb, or anti-CR3 mAb for 1 hour, and then were infected with (B) opsonized or (C) non-opsonized FITC-labeled wild-type or Δ choD strains for 2 hours. In all samples, macrophages with ingested bacteria were counted using a fluorescence microscope. All data are presented as the percentage of macrophages involved in phagocytosis, expressed as means ± SEMs (*p≤0.02, Mtb strain vs. Mtb strain+IRAK1/4 inhibitor or anti-TLR2 mAb or anti-CR3 mAb, # p≤0.03, Δ choD +anti-CR3 mAb vs. Δ choD +anti-TLR2 mAb,; Mann-Whitney U test). Data are presented from five independent experiments. Each experiment was carried out in duplicate.

Techniques Used: Infection, Labeling, Fluorescence, Microscopy, MANN-WHITNEY

ROS production by infected macrophages. (A) Macrophages were infected with wild-type, Δ choD , or Δ choD - choD strains for 2 hours, washed with HBSS, and then cultured for 24 hours. (B and C) Macrophages were incubated with IRAK1/4 inhibitor or anti-CR3 mAb for 1 hour, and then were infected with opsonized (B) or non-opsonized (C) wild-type or Δ choD strains for 2 hours, washed with HBSS, and cultured for 24 hours. Cells were then stimulated with PMA, and ROS production was assessed using the CL assay. Data are presented as the percentage inhibition of ROS production, expressed as means ± SEMs (*p≤0.04, Δ choD vs. wild-type or Δ choD - choD ; #p≤0.05, wild-type vs. wide-type+IRAK1/4 inhibitor; **p≤0.05, Δ choD vs. Δ choD +IRAK1/4 inhibitor; Mann-Whitney U test). Data are presented from five independent experiments. Every experiment was carried out in triplicate.
Figure Legend Snippet: ROS production by infected macrophages. (A) Macrophages were infected with wild-type, Δ choD , or Δ choD - choD strains for 2 hours, washed with HBSS, and then cultured for 24 hours. (B and C) Macrophages were incubated with IRAK1/4 inhibitor or anti-CR3 mAb for 1 hour, and then were infected with opsonized (B) or non-opsonized (C) wild-type or Δ choD strains for 2 hours, washed with HBSS, and cultured for 24 hours. Cells were then stimulated with PMA, and ROS production was assessed using the CL assay. Data are presented as the percentage inhibition of ROS production, expressed as means ± SEMs (*p≤0.04, Δ choD vs. wild-type or Δ choD - choD ; #p≤0.05, wild-type vs. wide-type+IRAK1/4 inhibitor; **p≤0.05, Δ choD vs. Δ choD +IRAK1/4 inhibitor; Mann-Whitney U test). Data are presented from five independent experiments. Every experiment was carried out in triplicate.

Techniques Used: Infection, Cell Culture, Incubation, Inhibition, MANN-WHITNEY

Survival of Mtb in macrophages. (A) Macrophages were infected with wild-type, Δ choD or Δ choD - choD strains for 2 hours without inhibitor or mAb and then washed with HBSS. (B and C) Macrophages were incubated with IRAK1/4 inhibitor, anti-TLR2 blocking mAb, or anti-CR3 blocking mAb for 1 hour prior to infection with opsonized (B) or non-opsonized (C) wild-type or Δ choD strains, and then washed. On the day of infection and after 6 days in culture, macrophages were lysed with Triton X-100 and cell lysates were plated onto agar plates. After 21 days of culture, CFUs were counted. The data are presented as fold increase in CFUs, expressed as means ± SEMs (*p≤0.03, Δ choD vs. wild-type or Δ choD - choD ; #p≤0.04, Δ choD vs. Δ choD +IRAK1/4 inhibitor or Δ choD +anti-TLR2 mAb or Δ choD +anti-CR3 mAb; Mann-Whitney U test). Data are presented from five independent experiments. Each experiment was carried out in duplicate.
Figure Legend Snippet: Survival of Mtb in macrophages. (A) Macrophages were infected with wild-type, Δ choD or Δ choD - choD strains for 2 hours without inhibitor or mAb and then washed with HBSS. (B and C) Macrophages were incubated with IRAK1/4 inhibitor, anti-TLR2 blocking mAb, or anti-CR3 blocking mAb for 1 hour prior to infection with opsonized (B) or non-opsonized (C) wild-type or Δ choD strains, and then washed. On the day of infection and after 6 days in culture, macrophages were lysed with Triton X-100 and cell lysates were plated onto agar plates. After 21 days of culture, CFUs were counted. The data are presented as fold increase in CFUs, expressed as means ± SEMs (*p≤0.03, Δ choD vs. wild-type or Δ choD - choD ; #p≤0.04, Δ choD vs. Δ choD +IRAK1/4 inhibitor or Δ choD +anti-TLR2 mAb or Δ choD +anti-CR3 mAb; Mann-Whitney U test). Data are presented from five independent experiments. Each experiment was carried out in duplicate.

Techniques Used: Infection, Incubation, Blocking Assay, MANN-WHITNEY

NO production by infected macrophages. (A) Macrophages were infected with wild-type, Δ choD , or Δ choD - choD strains for 2 hours without inhibitor or mAb and then washed with HBSS. (B and C) Macrophages were incubated with IRAK1/4 inhibitor or anti-CR3 blocking mAb for 1 hour prior to infection with opsonized (B) or non-opsonized (C) wild-type or Δ choD strains. After culturing for 48 hours, the concentration of nitrite, a stable metabolite of NO, was assessed in culture supernatants using the Griess reagent. The data are presented as nitrite concentration (µM), expressed as means ± SEMs (*p≤0.03, Mtb strain vs. none (macrophages in CM); Wilcoxon’s signed-rank test; #p≤0.04, Δ choD vs. Δ choD +IRAK1/4 inhibitor; Mann-Whitney U test). Neither IRAK1/4 inhibitor nor anti-CR3 mAb significantly influenced NO production by uninfected macrophages. Data are presented from six independent experiments. Each experiment was carried out in triplicate.
Figure Legend Snippet: NO production by infected macrophages. (A) Macrophages were infected with wild-type, Δ choD , or Δ choD - choD strains for 2 hours without inhibitor or mAb and then washed with HBSS. (B and C) Macrophages were incubated with IRAK1/4 inhibitor or anti-CR3 blocking mAb for 1 hour prior to infection with opsonized (B) or non-opsonized (C) wild-type or Δ choD strains. After culturing for 48 hours, the concentration of nitrite, a stable metabolite of NO, was assessed in culture supernatants using the Griess reagent. The data are presented as nitrite concentration (µM), expressed as means ± SEMs (*p≤0.03, Mtb strain vs. none (macrophages in CM); Wilcoxon’s signed-rank test; #p≤0.04, Δ choD vs. Δ choD +IRAK1/4 inhibitor; Mann-Whitney U test). Neither IRAK1/4 inhibitor nor anti-CR3 mAb significantly influenced NO production by uninfected macrophages. Data are presented from six independent experiments. Each experiment was carried out in triplicate.

Techniques Used: Infection, Incubation, Blocking Assay, Concentration Assay, MANN-WHITNEY

12) Product Images from "Mycobacterium avium subsp. paratuberculosis induces differential cytosine methylation at miR-21 transcription start site region"

Article Title: Mycobacterium avium subsp. paratuberculosis induces differential cytosine methylation at miR-21 transcription start site region

Journal: Iranian Journal of Veterinary Research

doi:

Ziehl-Neelsen staining of THP-1 macrophages infected with MAP. ( A ) THP-1 cell infected with MAP (arrows), and ( B ) Control cell culture (magnification ×100)
Figure Legend Snippet: Ziehl-Neelsen staining of THP-1 macrophages infected with MAP. ( A ) THP-1 cell infected with MAP (arrows), and ( B ) Control cell culture (magnification ×100)

Techniques Used: Staining, Infection, Cell Culture

Predicted binding sites for TFs known to be involved in miR-21 regulation are differentially methylated at the miR-21 TSS region in MAP infected THP-1 macrophages. Squares show the position of the CpG di-nucleotides in the amplified sequences analysed for differential methylation. Solid squares show differentially methylated cytosines (bolded in the TF binding sequences indicated in the boxes)
Figure Legend Snippet: Predicted binding sites for TFs known to be involved in miR-21 regulation are differentially methylated at the miR-21 TSS region in MAP infected THP-1 macrophages. Squares show the position of the CpG di-nucleotides in the amplified sequences analysed for differential methylation. Solid squares show differentially methylated cytosines (bolded in the TF binding sequences indicated in the boxes)

Techniques Used: Binding Assay, Methylation, Infection, Amplification

13) Product Images from "Novel Mechanisms of Myocardial Ischemia, Ischemia-Reperfusion, and Protection by Myocardial Conditioning: Infarct size-limiting effect of epoxyeicosatrienoic acid analog EET-B is mediated by hypoxia-inducible factor-1α via downregulation of prolyl hydroxylase 3"

Article Title: Novel Mechanisms of Myocardial Ischemia, Ischemia-Reperfusion, and Protection by Myocardial Conditioning: Infarct size-limiting effect of epoxyeicosatrienoic acid analog EET-B is mediated by hypoxia-inducible factor-1α via downregulation of prolyl hydroxylase 3

Journal: American Journal of Physiology - Heart and Circulatory Physiology

doi: 10.1152/ajpheart.00726.2017

Hypoxia-inducible factor (HIF)-1α tissue immunopositivity ( A ) and prolyl-4-hydroxylase domain protein 3 (PHD3) nuclear positivity ( B ) in the nonischemic septum assessed at the end of ischemia (ISCH) and after 20 min and 2 h of reperfusion (REP) in rats administrated vehicle (Veh) or the epoxyeicosatrienoic acid (EET) analog EET-B (2.5 mg/kg) 5 min before reperfusion. Values are means ± SE. For HIF-1α, 4 different sections (six images in each) of 3 hearts/group were analyzed; for PHD3, 25 nuclei from 4 different sections of 3 hearts/group were analyzed. * P
Figure Legend Snippet: Hypoxia-inducible factor (HIF)-1α tissue immunopositivity ( A ) and prolyl-4-hydroxylase domain protein 3 (PHD3) nuclear positivity ( B ) in the nonischemic septum assessed at the end of ischemia (ISCH) and after 20 min and 2 h of reperfusion (REP) in rats administrated vehicle (Veh) or the epoxyeicosatrienoic acid (EET) analog EET-B (2.5 mg/kg) 5 min before reperfusion. Values are means ± SE. For HIF-1α, 4 different sections (six images in each) of 3 hearts/group were analyzed; for PHD3, 25 nuclei from 4 different sections of 3 hearts/group were analyzed. * P

Techniques Used:

Representative photomicrographs of immunohistochemical staining showing the expression and localization of hypoxia-inducible factor (HIF)-1α (black arrows; A and B ) and prolyl-4-hydroxylase domain protein 3 (PHD3; black arrows; D and E ) in left ventricles subjected to 30 min of ischemia (ISCH) followed by 20 min or 2 h of reperfusion (REP) in rats administrated vehicle (Veh) or the epoxyeicosatrienoic acid (EET) analog EET-B (2.5 mg/kg) 5 min before reperfusion. Bottom photos show details from the boxed regions shown on the top photos. Scale bars = 50 μm. C and F : quantification of left ventricular myocardial HIF-1α ( C )- and PHD3 ( F )-positive areas assessed at the end of ischemia and after 20 min and 2 h of reperfusion. Values are means ± SE from 4 different sections (6 images in each) of 3 hearts/group. * P
Figure Legend Snippet: Representative photomicrographs of immunohistochemical staining showing the expression and localization of hypoxia-inducible factor (HIF)-1α (black arrows; A and B ) and prolyl-4-hydroxylase domain protein 3 (PHD3; black arrows; D and E ) in left ventricles subjected to 30 min of ischemia (ISCH) followed by 20 min or 2 h of reperfusion (REP) in rats administrated vehicle (Veh) or the epoxyeicosatrienoic acid (EET) analog EET-B (2.5 mg/kg) 5 min before reperfusion. Bottom photos show details from the boxed regions shown on the top photos. Scale bars = 50 μm. C and F : quantification of left ventricular myocardial HIF-1α ( C )- and PHD3 ( F )-positive areas assessed at the end of ischemia and after 20 min and 2 h of reperfusion. Values are means ± SE from 4 different sections (6 images in each) of 3 hearts/group. * P

Techniques Used: Immunohistochemistry, Staining, Expressing

14) Product Images from "Differentiation of adipose-derived stem cells into Schwann cell-like cells through intermittent induction: potential advantage of cellular transient memory function"

Article Title: Differentiation of adipose-derived stem cells into Schwann cell-like cells through intermittent induction: potential advantage of cellular transient memory function

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-018-0884-3

Identification of adipose-derived stem cells (ASCs) and schematic of the experimental design. a Primary ASCs grew in clusters and had a rounded spindle-like shape. b ASCs at passage 2 could differentiate into adipocytes, and the visual field in photographs was filled with red lipid droplets stained with Oil Red O solution. c ASCs differentiated into osteocytes formed calcium nodules with burrs, which were stained red by Alizarin Red solution. d In cartilage pellets, many cartilage lacunae were found, and the glycosaminoglycans around the chondrocytes were stained purple-blue by Toluidine Blue O solution. e Flow cytometry showed that more than 98% of ASCs were immunopositive for the MSC markers CD29, CD44, and CD90, while less than 6% of ASCs were immunopositive for the hematopoietic stem cell markers CD34, CD45, and CD86. f Schematic showing the experimental groups and induction methods. bFGF, basic fibroblast growth factor; β-ME, β-mercaptoethanol; dASC, differentiated ASC; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PDGF, platelet-derived growth factor; SC, Schwann cell; uASC, undifferentiated ASC
Figure Legend Snippet: Identification of adipose-derived stem cells (ASCs) and schematic of the experimental design. a Primary ASCs grew in clusters and had a rounded spindle-like shape. b ASCs at passage 2 could differentiate into adipocytes, and the visual field in photographs was filled with red lipid droplets stained with Oil Red O solution. c ASCs differentiated into osteocytes formed calcium nodules with burrs, which were stained red by Alizarin Red solution. d In cartilage pellets, many cartilage lacunae were found, and the glycosaminoglycans around the chondrocytes were stained purple-blue by Toluidine Blue O solution. e Flow cytometry showed that more than 98% of ASCs were immunopositive for the MSC markers CD29, CD44, and CD90, while less than 6% of ASCs were immunopositive for the hematopoietic stem cell markers CD34, CD45, and CD86. f Schematic showing the experimental groups and induction methods. bFGF, basic fibroblast growth factor; β-ME, β-mercaptoethanol; dASC, differentiated ASC; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PDGF, platelet-derived growth factor; SC, Schwann cell; uASC, undifferentiated ASC

Techniques Used: Derivative Assay, Staining, Flow Cytometry, Cytometry, Modification

15) Product Images from "Engagement of activated Notch signalling in collagen II-specific T helper type 1 (Th1)- and Th17-type expansion involving Notch3 and Delta-like1"

Article Title: Engagement of activated Notch signalling in collagen II-specific T helper type 1 (Th1)- and Th17-type expansion involving Notch3 and Delta-like1

Journal: Clinical and Experimental Immunology

doi: 10.1111/j.1365-2249.2010.04310.x

Collagen-specific reactivation tends to T helper type 1 (Th1)- and Th17-type expansion along with activated Notch signalling and increased Notch3 expression. (a) Spleen mononuclear cells (SMNCs) from collagen II (CII)-immunized DBA/1J mice were cultured in vitro with or without CII; 3 days later, cells were collected and the percentage of Th1, regulatory T cells (T reg ) and Th17 cells were analysed using flow cytometric intracellular staining, as described in Methods. (b) The representative flow cytometric results summarized in (a) are shown; the percentages of relative cytokine- or transcript factor-expression T cells are indicated in the dot-plots. (c) After 3 days' culture with or without CII, CD4 + T cells were purified from SMNCs by magnetic sorting kits and were assessed for transcript levels of Hes1 and four Notch receptors, including Notch1, Notch2, Notch3 and Notch4 by real-time polymerase chain reaction (PCR). * P
Figure Legend Snippet: Collagen-specific reactivation tends to T helper type 1 (Th1)- and Th17-type expansion along with activated Notch signalling and increased Notch3 expression. (a) Spleen mononuclear cells (SMNCs) from collagen II (CII)-immunized DBA/1J mice were cultured in vitro with or without CII; 3 days later, cells were collected and the percentage of Th1, regulatory T cells (T reg ) and Th17 cells were analysed using flow cytometric intracellular staining, as described in Methods. (b) The representative flow cytometric results summarized in (a) are shown; the percentages of relative cytokine- or transcript factor-expression T cells are indicated in the dot-plots. (c) After 3 days' culture with or without CII, CD4 + T cells were purified from SMNCs by magnetic sorting kits and were assessed for transcript levels of Hes1 and four Notch receptors, including Notch1, Notch2, Notch3 and Notch4 by real-time polymerase chain reaction (PCR). * P

Techniques Used: Expressing, Mouse Assay, Cell Culture, In Vitro, Flow Cytometry, Staining, Purification, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Inhibition of Notch signalling by N -[ N -(3,5-difluorophenacetyl)-L-alanyl]- S -phenylglycine t -butyl ester (DAPT) and Notch3 antibody decrease the collagen-specific T cell proliferation and attenuate T helper type 1 (Th1)- and Th17-type responses. Spleen mononuclear cells (SMNCs) from collagen II (CII)-immunized DBA/1J mice were restimulated with CII in the presence of DAPT (5 µM) or α-Notch3 (10 µg/ml). (a) The proliferation of SMNCs was determined by tritiated thymidine ( 3 [H]-TdR) incorporation. Results were expressed as mean ± standard deviation of counts per minute (cpm) of triplicates or quadruplicates. (b) The percentage of Th1, regulatory T cells (T reg ) and Th17 cells in SMNCs were analysed using flow cytometric intracellular staining.
Figure Legend Snippet: Inhibition of Notch signalling by N -[ N -(3,5-difluorophenacetyl)-L-alanyl]- S -phenylglycine t -butyl ester (DAPT) and Notch3 antibody decrease the collagen-specific T cell proliferation and attenuate T helper type 1 (Th1)- and Th17-type responses. Spleen mononuclear cells (SMNCs) from collagen II (CII)-immunized DBA/1J mice were restimulated with CII in the presence of DAPT (5 µM) or α-Notch3 (10 µg/ml). (a) The proliferation of SMNCs was determined by tritiated thymidine ( 3 [H]-TdR) incorporation. Results were expressed as mean ± standard deviation of counts per minute (cpm) of triplicates or quadruplicates. (b) The percentage of Th1, regulatory T cells (T reg ) and Th17 cells in SMNCs were analysed using flow cytometric intracellular staining.

Techniques Used: Inhibition, Mouse Assay, Standard Deviation, Flow Cytometry, Staining

16) Product Images from "A glutaredoxin in the mitochondrial intermembrane space has stage-specific functions in the thermo-tolerance and proliferation of African trypanosomes"

Article Title: A glutaredoxin in the mitochondrial intermembrane space has stage-specific functions in the thermo-tolerance and proliferation of African trypanosomes

Journal: Redox Biology

doi: 10.1016/j.redox.2018.01.011

Morphological analysis of Grx2-depleted PC T. brucei . A. Grx2 RNAi cells grown for three days in the absence (- tet) and presence of tet (+ tet) were stained with MitoTracker and DAPI and analyzed by fluorescence microscopy. Induction of RNAi leads to an elongated phenotype. The merge of phase contrast and DAPI images (upper panel) as well as the MitoTracker staining (lower panel) are shown for representative cells. Scale bar: 10 µm. B. Quantification of cellular distances in 210 WT parasites and 210 Grx2-RNAi cells induced for 3 days (+ tet) with 1K1N configuration, irrespective of the overall cell morphology. The minimum, mean, and maximum distances as well as the mean distance ± standard deviations are depicted. Distances were measured using ImageJ software. The p-values were calculated by Student's unpaired t -test (***, p-value ≤ .001). C. Karyotype analysis of WT, non-induced (- tet) and induced (+ tet) Grx2 RNAi cells. At least 280 cells were inspected for their kinetoplast and nucleus content. The percentage of cells with the respective configuration is given for two independent experiments. 1K*1N are cells with one elongated kinetoplast and one nucleus. Other indicates cells with abnormal N-K distribution.
Figure Legend Snippet: Morphological analysis of Grx2-depleted PC T. brucei . A. Grx2 RNAi cells grown for three days in the absence (- tet) and presence of tet (+ tet) were stained with MitoTracker and DAPI and analyzed by fluorescence microscopy. Induction of RNAi leads to an elongated phenotype. The merge of phase contrast and DAPI images (upper panel) as well as the MitoTracker staining (lower panel) are shown for representative cells. Scale bar: 10 µm. B. Quantification of cellular distances in 210 WT parasites and 210 Grx2-RNAi cells induced for 3 days (+ tet) with 1K1N configuration, irrespective of the overall cell morphology. The minimum, mean, and maximum distances as well as the mean distance ± standard deviations are depicted. Distances were measured using ImageJ software. The p-values were calculated by Student's unpaired t -test (***, p-value ≤ .001). C. Karyotype analysis of WT, non-induced (- tet) and induced (+ tet) Grx2 RNAi cells. At least 280 cells were inspected for their kinetoplast and nucleus content. The percentage of cells with the respective configuration is given for two independent experiments. 1K*1N are cells with one elongated kinetoplast and one nucleus. Other indicates cells with abnormal N-K distribution.

Techniques Used: Staining, Fluorescence, Microscopy, Software

17) Product Images from "Influence of Cyclooxygenase-2 Inhibitors on Kynurenic Acid Production in Rat Brain in Vitro"

Article Title: Influence of Cyclooxygenase-2 Inhibitors on Kynurenic Acid Production in Rat Brain in Vitro

Journal: Neurotoxicity Research

doi: 10.1007/s12640-018-9952-9

Binding pocket of parecoxib within the KAT II crystal structure. Two ligand orientations overlapping the KYN binding site within the KAT II (Han et al. 2008 ). Ligand at orientation 1 (yellow) and orientation 2 (magenta) is presented with co-factor; PMP (orange) all rendered in stick mode; KAT II molecular surface is shown in gray. Non-polar hydrogen atoms are hidden
Figure Legend Snippet: Binding pocket of parecoxib within the KAT II crystal structure. Two ligand orientations overlapping the KYN binding site within the KAT II (Han et al. 2008 ). Ligand at orientation 1 (yellow) and orientation 2 (magenta) is presented with co-factor; PMP (orange) all rendered in stick mode; KAT II molecular surface is shown in gray. Non-polar hydrogen atoms are hidden

Techniques Used: Binding Assay

Molecular structures of niflumic acid, parecoxib, celecoxib, and KYN—physiological substrate of KAT II
Figure Legend Snippet: Molecular structures of niflumic acid, parecoxib, celecoxib, and KYN—physiological substrate of KAT II

Techniques Used:

Influence of celecoxib ( a ), niflumic acid ( b ), and parecoxib ( c ) on KAT II activity in rat brain cortex in vitro. Data are expressed as mean percentage of control KYNA production ± SD, n = 3, Kruskal-Wallis with Dunn’s post hoc test, ** P
Figure Legend Snippet: Influence of celecoxib ( a ), niflumic acid ( b ), and parecoxib ( c ) on KAT II activity in rat brain cortex in vitro. Data are expressed as mean percentage of control KYNA production ± SD, n = 3, Kruskal-Wallis with Dunn’s post hoc test, ** P

Techniques Used: Activity Assay, In Vitro

Influence of celecoxib ( a ), niflumic acid ( b ), and parecoxib ( c ) on KYNA production in rat brain cortical slices in vitro. Data are expressed as mean percentage of KYNA production ± SD, n = 6, Kruskal-Wallis with Dunn’s post hoc test, ** P
Figure Legend Snippet: Influence of celecoxib ( a ), niflumic acid ( b ), and parecoxib ( c ) on KYNA production in rat brain cortical slices in vitro. Data are expressed as mean percentage of KYNA production ± SD, n = 6, Kruskal-Wallis with Dunn’s post hoc test, ** P

Techniques Used: In Vitro

18) Product Images from "Isolation, characterization, and functional expression of kynurenine aminotransferase cDNA from the yellow fever mosquito, Aedes aegypti"

Article Title: Isolation, characterization, and functional expression of kynurenine aminotransferase cDNA from the yellow fever mosquito, Aedes aegypti

Journal: Insect biochemistry and molecular biology

doi:

Specific KAT activity of crude larval, pupal and adult protein. Protein from 4-day-old larvae, 12 h pupae and 3-day-old adult mosquitoes was extracted as described in Section 2. The reaction mixture of 100 μl consisting of 10 mM kynurenine or 5 mM 3-HK, 70 μM PLP, 12 mM pyruvate and 0.5 mg of crude larval, pupal or adult protein was prepared in 100 mM tris buffer (pH 8.5) and incubated at 50°C for 10 min. KA or XA produced in the corresponding reaction mixtures was quantified by HPLC-UV analysis. Solid bar and open bar illustrate the specific activity of crude larval (L), pupal (P) and adult (A) protein to kynurenine and 3-HK, respectively.
Figure Legend Snippet: Specific KAT activity of crude larval, pupal and adult protein. Protein from 4-day-old larvae, 12 h pupae and 3-day-old adult mosquitoes was extracted as described in Section 2. The reaction mixture of 100 μl consisting of 10 mM kynurenine or 5 mM 3-HK, 70 μM PLP, 12 mM pyruvate and 0.5 mg of crude larval, pupal or adult protein was prepared in 100 mM tris buffer (pH 8.5) and incubated at 50°C for 10 min. KA or XA produced in the corresponding reaction mixtures was quantified by HPLC-UV analysis. Solid bar and open bar illustrate the specific activity of crude larval (L), pupal (P) and adult (A) protein to kynurenine and 3-HK, respectively.

Techniques Used: Activity Assay, Plasmid Purification, Incubation, Produced, High Performance Liquid Chromatography

19) Product Images from "An Electrochemical Fiveplex Biochip Assay Based on Anti-Idiotypic Antibodies for Fast On-Site Detection of Bioterrorism Relevant Low Molecular Weight Toxins"

Article Title: An Electrochemical Fiveplex Biochip Assay Based on Anti-Idiotypic Antibodies for Fast On-Site Detection of Bioterrorism Relevant Low Molecular Weight Toxins

Journal: Toxins

doi: 10.3390/toxins11120696

Schematic procedure of the automated fiveplex anti-idiotypic antibody-based competitive immunoassay for detection of STX, MC-LR, T-2, RoA and AFB1 upon an electrochemical biochip. ( 1 ) Application of the sample mixed with monoclonal detection antibody-β-D-galactosidase (mAb-bGAL) tracer cocktail to the biochip pre-coated with capture mAbs; ( 2 ) Competition reaction upon the biochip; ( 3 ) bGAL substrate flow as well as amperometric readout of each electrode position. (Note: Illustrated biochip scheme represents only a small section of the entire electrode positions).
Figure Legend Snippet: Schematic procedure of the automated fiveplex anti-idiotypic antibody-based competitive immunoassay for detection of STX, MC-LR, T-2, RoA and AFB1 upon an electrochemical biochip. ( 1 ) Application of the sample mixed with monoclonal detection antibody-β-D-galactosidase (mAb-bGAL) tracer cocktail to the biochip pre-coated with capture mAbs; ( 2 ) Competition reaction upon the biochip; ( 3 ) bGAL substrate flow as well as amperometric readout of each electrode position. (Note: Illustrated biochip scheme represents only a small section of the entire electrode positions).

Techniques Used: Flow Cytometry

20) Product Images from "Lethal myelofibrosis induced by Bmi1-deficient hematopoietic cells unveils a tumor suppressor function of the polycomb group genes"

Article Title: Lethal myelofibrosis induced by Bmi1-deficient hematopoietic cells unveils a tumor suppressor function of the polycomb group genes

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20111709

Forced expression of Hmga2 causes myeloproliferative hematopoiesis with enhanced megakaryocytopoiesis. (A) Donor chimerism and lineage contribution in PB ( n = 5). CD34 − LSK HSCs (CD45.2) transduced with either control or Hmga2 retrovirus were transplanted into lethally irradiated CD45.1 mice together with 2 × 10 5 BM competitor cells from 8-wk-old CD45.1 mice. The chimerism of CD45.2 + GFP + -transduced cells in PB of recipient mice was examined at 6 mo after transplantation (left). Lineage contribution of CD45.2 + GFP + donor cells to myeloid (Gr-1 + and/or Mac-1 + ), B (B220 + ), or T (CD4 + or CD8 + ) cells (right). Data are presented as the mean ± SD ( n = 6). (B) Absolute numbers of BM mononuclear cells (from bilateral femurs and tibiae) and spleen weights at 6 mo after transplantation. Data are presented as the mean ± SD ( n = 6). *, P
Figure Legend Snippet: Forced expression of Hmga2 causes myeloproliferative hematopoiesis with enhanced megakaryocytopoiesis. (A) Donor chimerism and lineage contribution in PB ( n = 5). CD34 − LSK HSCs (CD45.2) transduced with either control or Hmga2 retrovirus were transplanted into lethally irradiated CD45.1 mice together with 2 × 10 5 BM competitor cells from 8-wk-old CD45.1 mice. The chimerism of CD45.2 + GFP + -transduced cells in PB of recipient mice was examined at 6 mo after transplantation (left). Lineage contribution of CD45.2 + GFP + donor cells to myeloid (Gr-1 + and/or Mac-1 + ), B (B220 + ), or T (CD4 + or CD8 + ) cells (right). Data are presented as the mean ± SD ( n = 6). (B) Absolute numbers of BM mononuclear cells (from bilateral femurs and tibiae) and spleen weights at 6 mo after transplantation. Data are presented as the mean ± SD ( n = 6). *, P

Techniques Used: Expressing, Transduction, Irradiation, Mouse Assay, Transplantation Assay

Hmga2 promotes expansion of progenitor cells and enhances megakaryocytopoiesis in vitro. (A) Growth of CD34 − LSK HSCs transduced with the Hmga2 retrovirus. 100 CD34 − LSK HSCs were transduced with either the empty vector or Hmga2 retrovirus and cultured in the presence of SCF, TPO, IL-3, FP6, and EPO for 14 d. Numbers of cells are shown as the mean ± SD for triplicate cultures. **, P
Figure Legend Snippet: Hmga2 promotes expansion of progenitor cells and enhances megakaryocytopoiesis in vitro. (A) Growth of CD34 − LSK HSCs transduced with the Hmga2 retrovirus. 100 CD34 − LSK HSCs were transduced with either the empty vector or Hmga2 retrovirus and cultured in the presence of SCF, TPO, IL-3, FP6, and EPO for 14 d. Numbers of cells are shown as the mean ± SD for triplicate cultures. **, P

Techniques Used: In Vitro, Transduction, Plasmid Preparation, Cell Culture

21) Product Images from "Sandwich Electrochemical Immunosensor for Early Detection of Tuberculosis Based on Graphene/Polyaniline-Modified Screen-Printed Gold Electrode"

Article Title: Sandwich Electrochemical Immunosensor for Early Detection of Tuberculosis Based on Graphene/Polyaniline-Modified Screen-Printed Gold Electrode

Journal: Sensors (Basel, Switzerland)

doi: 10.3390/s18113926

FESEM images of ( a ) graphene and ( b ) GP/PANI nanocomposite with magnification of 50k. ( c ) IR spectra of GP nanosheet and GP/PANI nanocomposite.
Figure Legend Snippet: FESEM images of ( a ) graphene and ( b ) GP/PANI nanocomposite with magnification of 50k. ( c ) IR spectra of GP nanosheet and GP/PANI nanocomposite.

Techniques Used:

22) Product Images from "p53 and p73 Regulate Apoptosis but Not Cell-Cycle Progression in Mouse Embryonic Stem Cells upon DNA Damage and Differentiation"

Article Title: p53 and p73 Regulate Apoptosis but Not Cell-Cycle Progression in Mouse Embryonic Stem Cells upon DNA Damage and Differentiation

Journal: Stem Cell Reports

doi: 10.1016/j.stemcr.2016.10.008

Differentiation-Induced Apoptosis of R1 Cells (A) R1 cells were cultured in the absence of LIF for 72 hr followed by AP staining. Representative images were captured by phase-contrast microscopy. (B) R1 cells were cultured in the absence of LIF for 72 or 96 hr. Cells were then subjected to western blot analyses. β-Actin served as a loading control. (C and D) R1 sh-GFP, R1 sh-p53-1, R1 sh-p53-2, R1 sh-p73-1, R1 sh-p73-2, and R1 sh-p53-1/sh-p73-1 cells were cultured in the absence of LIF for 72 or 96 hr. Cells were subjected to western blot (C) or FACS analyses (D). At least three independent experiments were performed. Representative histograms and western blot data are shown. See also Figure S5 .
Figure Legend Snippet: Differentiation-Induced Apoptosis of R1 Cells (A) R1 cells were cultured in the absence of LIF for 72 hr followed by AP staining. Representative images were captured by phase-contrast microscopy. (B) R1 cells were cultured in the absence of LIF for 72 or 96 hr. Cells were then subjected to western blot analyses. β-Actin served as a loading control. (C and D) R1 sh-GFP, R1 sh-p53-1, R1 sh-p53-2, R1 sh-p73-1, R1 sh-p73-2, and R1 sh-p53-1/sh-p73-1 cells were cultured in the absence of LIF for 72 or 96 hr. Cells were subjected to western blot (C) or FACS analyses (D). At least three independent experiments were performed. Representative histograms and western blot data are shown. See also Figure S5 .

Techniques Used: Cell Culture, Staining, Microscopy, Western Blot, FACS

23) Product Images from "Janus and PI3-kinases mediate glucocorticoid resistance in activated chronic leukemia cells"

Article Title: Janus and PI3-kinases mediate glucocorticoid resistance in activated chronic leukemia cells

Journal: Oncotarget

doi: 10.18632/oncotarget.11618

Effect of Ruxolitinib on DEX-mediated death in stimulated CLL cells in vitro and in vivo Purified CLL cells were stimulated with IL2 and Resiquimod in the presence or absence of dexamethasone (30 μM) or ruxolitinib (0.5 μM). a. Size changes of viable 7AAD- cells (median of forward scatter parameter (mfi)) were determined by flow cytometry after 48 h. Decreased size due to activation in the presence of ruxolitinib (RUX) is indicated for each patient sample by the lines. b. After 48 h, levels of TNFα (left panel, n = 5) and IL10 (right panel, n = 11) in the culture supernatants of 2S cells in the presence and absence (CTR) of RUX were measured by ELISAs. c. After 48 h, specific death was calculated from the difference between the percentages of 7AAD- cells in control and DEX-treated samples 48 h after RUX treatment ( n = 12). d. Changes in circulating lymphocytes (WBC) as a measure of clinical response are shown for 3 patients 1 month after treatment with high-dose glucocorticoids in the presence or absence of Ruxolitinib at the doses indicated in the graphs. *, p
Figure Legend Snippet: Effect of Ruxolitinib on DEX-mediated death in stimulated CLL cells in vitro and in vivo Purified CLL cells were stimulated with IL2 and Resiquimod in the presence or absence of dexamethasone (30 μM) or ruxolitinib (0.5 μM). a. Size changes of viable 7AAD- cells (median of forward scatter parameter (mfi)) were determined by flow cytometry after 48 h. Decreased size due to activation in the presence of ruxolitinib (RUX) is indicated for each patient sample by the lines. b. After 48 h, levels of TNFα (left panel, n = 5) and IL10 (right panel, n = 11) in the culture supernatants of 2S cells in the presence and absence (CTR) of RUX were measured by ELISAs. c. After 48 h, specific death was calculated from the difference between the percentages of 7AAD- cells in control and DEX-treated samples 48 h after RUX treatment ( n = 12). d. Changes in circulating lymphocytes (WBC) as a measure of clinical response are shown for 3 patients 1 month after treatment with high-dose glucocorticoids in the presence or absence of Ruxolitinib at the doses indicated in the graphs. *, p

Techniques Used: In Vitro, In Vivo, Purification, Flow Cytometry, Cytometry, Activation Assay

24) Product Images from "The structure of the nucleoprotein of Influenza D shows that all Orthomyxoviridae nucleoproteins have a similar NPCORE, with or without a NPTAIL for nuclear transport"

Article Title: The structure of the nucleoprotein of Influenza D shows that all Orthomyxoviridae nucleoproteins have a similar NPCORE, with or without a NPTAIL for nuclear transport

Journal: Scientific Reports

doi: 10.1038/s41598-018-37306-y

Purification and characterized of Influenza D nucleoprotein. ( a ) Size exclusion chromatography profile of wild-type D/NP. The sample was loaded on a Hiload TM 16/600 S200 column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 300 mM NaCl and 5 mM β-mercaptoethanol. ( b ) SEC-MALLS-RI analysis of D/NP. SEC was performed with a Superdex TM 200 increase 10/300 GL column equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5 mM β-ME. The panel shows the theoretical Mw and the measured Mw. ( c ) and ( e ) Electron microscopy images of the elution peak of D/NP and D/NP-511. Samples show different oligomeric states although most oligomers are tetramers. The scale bar corresponds to 100 nm. ( d ) Coomassie blue-stained SDS-PAGE (4–20% gradient polyacrylamide) showing the purified wild-type D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511).
Figure Legend Snippet: Purification and characterized of Influenza D nucleoprotein. ( a ) Size exclusion chromatography profile of wild-type D/NP. The sample was loaded on a Hiload TM 16/600 S200 column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 300 mM NaCl and 5 mM β-mercaptoethanol. ( b ) SEC-MALLS-RI analysis of D/NP. SEC was performed with a Superdex TM 200 increase 10/300 GL column equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5 mM β-ME. The panel shows the theoretical Mw and the measured Mw. ( c ) and ( e ) Electron microscopy images of the elution peak of D/NP and D/NP-511. Samples show different oligomeric states although most oligomers are tetramers. The scale bar corresponds to 100 nm. ( d ) Coomassie blue-stained SDS-PAGE (4–20% gradient polyacrylamide) showing the purified wild-type D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511).

Techniques Used: Purification, Size-exclusion Chromatography, Electron Microscopy, Staining, SDS Page

Interaction of D/NP and D/NP TAIL with importin-α7. ( a ) Size exclusion chromatography profile of a mixture between human importin-α7 and D/NP TAIL . The mixture (molar ratio 1 importin-α7 for 2 D/NP TAIL ) was incubated 1 hour at room temperature and then loaded on a Superdex TM 75 10/300GL column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM β-mercaptoethanol. ( b ) Thermal stability assay of importin-α7 in absence (green) or in presence (red) of D/NP TAIL . In presence of D/NP TAIL , the melting temperature of importin-α7 is 5 °C higher. D/NP TAIL alone using Thermofluor did not give any denaturation signal (yellow curve). The upper insert corresponds to the derivative of the fluorescence signal for a precise measure of the melting temperature. ( c ) Affinity of importin-α7 for D/NP TAIL by measured by surface plasmon resonance (SPR). Biotinylated D/NP TAIL (left) and control peptide (right) were captured on a streptavidin-coated sensor chip surface before injections of several importin-α7 concentrations (10 nM in red, 25 nM in orange, 50 nM in green, 75 nM in blue and 100 nM in purple). The sensorgrams of the interaction between D/NP TAIL and importin-α7 were fitted under a Langmuir 1:1 binding model with mass-transfer (black line). ( d ) SEC-MALLS analysis of D/NP in complex with importin-α7. The mixture (molar ratio 1 D/NP for 1.2 importin-α7) was incubated 1 hour at room temperature and then loaded on a Superdex TM 200 increase 10/300 GL. The experimental molecular weight is consistent with the expected mass of four importins-α7 bound per D/NP tetramer. ( e ) Pull-down assays of human importin-α7 by D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511). The his-tags are on D/NP. The mixtures (molar ratio 1 D/NP for 1.2 importin-α7) were incubated 1 hour and the experience was done as described in panel ( a ). The figure shows the coomassie blue-stained SDS-PAGE (12% polyacrylamide) with the Load, FlowThough, Wash and the second fractions (E2).
Figure Legend Snippet: Interaction of D/NP and D/NP TAIL with importin-α7. ( a ) Size exclusion chromatography profile of a mixture between human importin-α7 and D/NP TAIL . The mixture (molar ratio 1 importin-α7 for 2 D/NP TAIL ) was incubated 1 hour at room temperature and then loaded on a Superdex TM 75 10/300GL column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM β-mercaptoethanol. ( b ) Thermal stability assay of importin-α7 in absence (green) or in presence (red) of D/NP TAIL . In presence of D/NP TAIL , the melting temperature of importin-α7 is 5 °C higher. D/NP TAIL alone using Thermofluor did not give any denaturation signal (yellow curve). The upper insert corresponds to the derivative of the fluorescence signal for a precise measure of the melting temperature. ( c ) Affinity of importin-α7 for D/NP TAIL by measured by surface plasmon resonance (SPR). Biotinylated D/NP TAIL (left) and control peptide (right) were captured on a streptavidin-coated sensor chip surface before injections of several importin-α7 concentrations (10 nM in red, 25 nM in orange, 50 nM in green, 75 nM in blue and 100 nM in purple). The sensorgrams of the interaction between D/NP TAIL and importin-α7 were fitted under a Langmuir 1:1 binding model with mass-transfer (black line). ( d ) SEC-MALLS analysis of D/NP in complex with importin-α7. The mixture (molar ratio 1 D/NP for 1.2 importin-α7) was incubated 1 hour at room temperature and then loaded on a Superdex TM 200 increase 10/300 GL. The experimental molecular weight is consistent with the expected mass of four importins-α7 bound per D/NP tetramer. ( e ) Pull-down assays of human importin-α7 by D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511). The his-tags are on D/NP. The mixtures (molar ratio 1 D/NP for 1.2 importin-α7) were incubated 1 hour and the experience was done as described in panel ( a ). The figure shows the coomassie blue-stained SDS-PAGE (12% polyacrylamide) with the Load, FlowThough, Wash and the second fractions (E2).

Techniques Used: Size-exclusion Chromatography, Incubation, Stability Assay, Fluorescence, SPR Assay, Chromatin Immunoprecipitation, Binding Assay, Molecular Weight, Staining, SDS Page

25) Product Images from "The structure of the nucleoprotein of Influenza D shows that all Orthomyxoviridae nucleoproteins have a similar NPCORE, with or without a NPTAIL for nuclear transport"

Article Title: The structure of the nucleoprotein of Influenza D shows that all Orthomyxoviridae nucleoproteins have a similar NPCORE, with or without a NPTAIL for nuclear transport

Journal: Scientific Reports

doi: 10.1038/s41598-018-37306-y

Purification and characterized of Influenza D nucleoprotein. ( a ) Size exclusion chromatography profile of wild-type D/NP. The sample was loaded on a Hiload TM 16/600 S200 column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 300 mM NaCl and 5 mM β-mercaptoethanol. ( b ) SEC-MALLS-RI analysis of D/NP. SEC was performed with a Superdex TM 200 increase 10/300 GL column equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5 mM β-ME. The panel shows the theoretical Mw and the measured Mw. ( c ) and ( e ) Electron microscopy images of the elution peak of D/NP and D/NP-511. Samples show different oligomeric states although most oligomers are tetramers. The scale bar corresponds to 100 nm. ( d ) Coomassie blue-stained SDS-PAGE (4–20% gradient polyacrylamide) showing the purified wild-type D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511).
Figure Legend Snippet: Purification and characterized of Influenza D nucleoprotein. ( a ) Size exclusion chromatography profile of wild-type D/NP. The sample was loaded on a Hiload TM 16/600 S200 column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 300 mM NaCl and 5 mM β-mercaptoethanol. ( b ) SEC-MALLS-RI analysis of D/NP. SEC was performed with a Superdex TM 200 increase 10/300 GL column equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5 mM β-ME. The panel shows the theoretical Mw and the measured Mw. ( c ) and ( e ) Electron microscopy images of the elution peak of D/NP and D/NP-511. Samples show different oligomeric states although most oligomers are tetramers. The scale bar corresponds to 100 nm. ( d ) Coomassie blue-stained SDS-PAGE (4–20% gradient polyacrylamide) showing the purified wild-type D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511).

Techniques Used: Purification, Size-exclusion Chromatography, Electron Microscopy, Staining, SDS Page

Interaction of D/NP and D/NP TAIL with importin-α7. ( a ) Size exclusion chromatography profile of a mixture between human importin-α7 and D/NP TAIL . The mixture (molar ratio 1 importin-α7 for 2 D/NP TAIL ) was incubated 1 hour at room temperature and then loaded on a Superdex TM 75 10/300GL column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM β-mercaptoethanol. ( b ) Thermal stability assay of importin-α7 in absence (green) or in presence (red) of D/NP TAIL . In presence of D/NP TAIL , the melting temperature of importin-α7 is 5 °C higher. D/NP TAIL alone using Thermofluor did not give any denaturation signal (yellow curve). The upper insert corresponds to the derivative of the fluorescence signal for a precise measure of the melting temperature. ( c ) Affinity of importin-α7 for D/NP TAIL by measured by surface plasmon resonance (SPR). Biotinylated D/NP TAIL (left) and control peptide (right) were captured on a streptavidin-coated sensor chip surface before injections of several importin-α7 concentrations (10 nM in red, 25 nM in orange, 50 nM in green, 75 nM in blue and 100 nM in purple). The sensorgrams of the interaction between D/NP TAIL and importin-α7 were fitted under a Langmuir 1:1 binding model with mass-transfer (black line). ( d ) SEC-MALLS analysis of D/NP in complex with importin-α7. The mixture (molar ratio 1 D/NP for 1.2 importin-α7) was incubated 1 hour at room temperature and then loaded on a Superdex TM 200 increase 10/300 GL. The experimental molecular weight is consistent with the expected mass of four importins-α7 bound per D/NP tetramer. ( e ) Pull-down assays of human importin-α7 by D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511). The his-tags are on D/NP. The mixtures (molar ratio 1 D/NP for 1.2 importin-α7) were incubated 1 hour and the experience was done as described in panel ( a ). The figure shows the coomassie blue-stained SDS-PAGE (12% polyacrylamide) with the Load, FlowThough, Wash and the second fractions (E2).
Figure Legend Snippet: Interaction of D/NP and D/NP TAIL with importin-α7. ( a ) Size exclusion chromatography profile of a mixture between human importin-α7 and D/NP TAIL . The mixture (molar ratio 1 importin-α7 for 2 D/NP TAIL ) was incubated 1 hour at room temperature and then loaded on a Superdex TM 75 10/300GL column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM β-mercaptoethanol. ( b ) Thermal stability assay of importin-α7 in absence (green) or in presence (red) of D/NP TAIL . In presence of D/NP TAIL , the melting temperature of importin-α7 is 5 °C higher. D/NP TAIL alone using Thermofluor did not give any denaturation signal (yellow curve). The upper insert corresponds to the derivative of the fluorescence signal for a precise measure of the melting temperature. ( c ) Affinity of importin-α7 for D/NP TAIL by measured by surface plasmon resonance (SPR). Biotinylated D/NP TAIL (left) and control peptide (right) were captured on a streptavidin-coated sensor chip surface before injections of several importin-α7 concentrations (10 nM in red, 25 nM in orange, 50 nM in green, 75 nM in blue and 100 nM in purple). The sensorgrams of the interaction between D/NP TAIL and importin-α7 were fitted under a Langmuir 1:1 binding model with mass-transfer (black line). ( d ) SEC-MALLS analysis of D/NP in complex with importin-α7. The mixture (molar ratio 1 D/NP for 1.2 importin-α7) was incubated 1 hour at room temperature and then loaded on a Superdex TM 200 increase 10/300 GL. The experimental molecular weight is consistent with the expected mass of four importins-α7 bound per D/NP tetramer. ( e ) Pull-down assays of human importin-α7 by D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511). The his-tags are on D/NP. The mixtures (molar ratio 1 D/NP for 1.2 importin-α7) were incubated 1 hour and the experience was done as described in panel ( a ). The figure shows the coomassie blue-stained SDS-PAGE (12% polyacrylamide) with the Load, FlowThough, Wash and the second fractions (E2).

Techniques Used: Size-exclusion Chromatography, Incubation, Stability Assay, Fluorescence, SPR Assay, Chromatin Immunoprecipitation, Binding Assay, Molecular Weight, Staining, SDS Page

26) Product Images from "Recognition of prostate-specific antigenic peptide determinants by human CD4 and CD8 T cells"

Article Title: Recognition of prostate-specific antigenic peptide determinants by human CD4 and CD8 T cells

Journal: Clinical and Experimental Immunology

doi: 10.1046/j.1365-2249.1998.00678.x

CD8 T cells specific for peptides of PSA and PSM in HLA-A1 individuals
Figure Legend Snippet: CD8 T cells specific for peptides of PSA and PSM in HLA-A1 individuals

Techniques Used:

27) Product Images from "Expanded potential stem cell media as a tool to study human developmental hematopoiesis in vitro"

Article Title: Expanded potential stem cell media as a tool to study human developmental hematopoiesis in vitro

Journal: Experimental Hematology

doi: 10.1016/j.exphem.2019.07.003

EPSCM PSC differentiation models human hematopoiesis in vitro. ( A ) Schematic of EPSCM PSC differentiation protocol. Human PSCs cultured in EPSCM were dissociated into a single cell suspension for EB formation in basal media (BM) consisting of DMEM/F-12 plus 20% KSR supplemented with ROCK inhibitor (ROCKi) at 1 × 10 5 cell/mL. After 48 hours, EBs were collected and transferred into human differentiation media (HDM) consisting of Knockout-DMEM plus 20% FBS, transferrin, ascorbic acid, and 2-mercaptoethanol. ( B ) Example image of EBs generated from EPSCM-maintained PSCs. ( C ) Percentage of KDR + cells within day 4 EBs generated from PB-EPSCM, EC-EPSCM, and H1-EPSCM lines using the differentiation strategy described in A. Each dot represents an individual differentiation ( n = 8). ( D ) Example flow cytometry plot displaying expression of KDR and CD140a in day 4 EBs, for differentiations described in B. ( E ) Percentage of CD43 + cells within day 14 EBs generated from PB-EPSCM, EC-EPSCM, and H1-EPSCM lines using the differentiation strategy described in A. Each dot represents an individual differentiation ( n = 14). ( F ) Example flow cytometry plot displaying expression of CD43 and CD34 (left), CD41 and CD45 (middle), and CD41 and CD235a (right) in day 14 EBs, for differentiations described in E. ( G ) Percentage of CD43 + subsets between EB day 4 and 20, derived from EC-EPSCM cells using the method described in A. Representative of two biological replicates. ( H ) Percentage of CD43 + subsets between EB day 4 and 20, derived from H1-EPSCM cells using the method described in A. Representative of two biological replicates. ( I ) Average number of CFUs from 50,000 EC-EPSCM-derived EB cells between day 8 and 20. Average of triplicates ± standard deviation. Representative of two biological replicates. ( J ) Average number of CFUs from 50,000 H1-EPSCM–derived EB cells at day 14 and 16. Average of triplicates ± standard deviation. Representative of two biological replicates.
Figure Legend Snippet: EPSCM PSC differentiation models human hematopoiesis in vitro. ( A ) Schematic of EPSCM PSC differentiation protocol. Human PSCs cultured in EPSCM were dissociated into a single cell suspension for EB formation in basal media (BM) consisting of DMEM/F-12 plus 20% KSR supplemented with ROCK inhibitor (ROCKi) at 1 × 10 5 cell/mL. After 48 hours, EBs were collected and transferred into human differentiation media (HDM) consisting of Knockout-DMEM plus 20% FBS, transferrin, ascorbic acid, and 2-mercaptoethanol. ( B ) Example image of EBs generated from EPSCM-maintained PSCs. ( C ) Percentage of KDR + cells within day 4 EBs generated from PB-EPSCM, EC-EPSCM, and H1-EPSCM lines using the differentiation strategy described in A. Each dot represents an individual differentiation ( n = 8). ( D ) Example flow cytometry plot displaying expression of KDR and CD140a in day 4 EBs, for differentiations described in B. ( E ) Percentage of CD43 + cells within day 14 EBs generated from PB-EPSCM, EC-EPSCM, and H1-EPSCM lines using the differentiation strategy described in A. Each dot represents an individual differentiation ( n = 14). ( F ) Example flow cytometry plot displaying expression of CD43 and CD34 (left), CD41 and CD45 (middle), and CD41 and CD235a (right) in day 14 EBs, for differentiations described in E. ( G ) Percentage of CD43 + subsets between EB day 4 and 20, derived from EC-EPSCM cells using the method described in A. Representative of two biological replicates. ( H ) Percentage of CD43 + subsets between EB day 4 and 20, derived from H1-EPSCM cells using the method described in A. Representative of two biological replicates. ( I ) Average number of CFUs from 50,000 EC-EPSCM-derived EB cells between day 8 and 20. Average of triplicates ± standard deviation. Representative of two biological replicates. ( J ) Average number of CFUs from 50,000 H1-EPSCM–derived EB cells at day 14 and 16. Average of triplicates ± standard deviation. Representative of two biological replicates.

Techniques Used: In Vitro, Cell Culture, Knock-Out, Generated, Flow Cytometry, Cytometry, Expressing, Derivative Assay, Standard Deviation

28) Product Images from "Structural basis of chimpanzee APOBEC3H dimerization stabilized by double-stranded RNA"

Article Title: Structural basis of chimpanzee APOBEC3H dimerization stabilized by double-stranded RNA

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky676

Effect of cpzA3H mutations in the RNA recognition interface on the intracellular levels of cpzA3H. WT cpzA3H and mutants were transiently expressed in 293T cells in the absence of an inhibitor or in the presence of MG132 (8 μM), chloroquine (100 μM) or bafilomycin A1 (80 nM). The proteins were detected by western blotting with the anti-DYKDDDDK tag monoclonal antibody (Anti-FLAG) or anti-β-tubulin antibody (Anti-β-Tubulin).
Figure Legend Snippet: Effect of cpzA3H mutations in the RNA recognition interface on the intracellular levels of cpzA3H. WT cpzA3H and mutants were transiently expressed in 293T cells in the absence of an inhibitor or in the presence of MG132 (8 μM), chloroquine (100 μM) or bafilomycin A1 (80 nM). The proteins were detected by western blotting with the anti-DYKDDDDK tag monoclonal antibody (Anti-FLAG) or anti-β-tubulin antibody (Anti-β-Tubulin).

Techniques Used: Western Blot

29) Product Images from "Development and Validation of an HPLC Method for Determination of Amifostine and/or Its Metabolite (WR-1065) In Human Plasma Using OPA Derivatization and UV Detection"

Article Title: Development and Validation of an HPLC Method for Determination of Amifostine and/or Its Metabolite (WR-1065) In Human Plasma Using OPA Derivatization and UV Detection

Journal: Iranian Journal of Pharmaceutical Research : IJPR

doi:

Alkylation of WR-1065 with IAA followed by derivatization with OPA-2-ME
Figure Legend Snippet: Alkylation of WR-1065 with IAA followed by derivatization with OPA-2-ME

Techniques Used:

Chromatograms of blank plasma reacted with OPA/2-ME reagent (larger box); blank plasma spiked with 30 µgmL −1 AMF, 50µgmL −1 WR-1065 and 30 µgmL −1 L-cysteine (as internal standard, IS) (box A); and blank plasma spiked with 50µgmL −1 WR-1065 and 30 µgmL −1 L-cysteine (box B).
Figure Legend Snippet: Chromatograms of blank plasma reacted with OPA/2-ME reagent (larger box); blank plasma spiked with 30 µgmL −1 AMF, 50µgmL −1 WR-1065 and 30 µgmL −1 L-cysteine (as internal standard, IS) (box A); and blank plasma spiked with 50µgmL −1 WR-1065 and 30 µgmL −1 L-cysteine (box B).

Techniques Used:

30) Product Images from "Rational Modification of Estrogen Receptor by Combination of Computational and Experimental Analysis"

Article Title: Rational Modification of Estrogen Receptor by Combination of Computational and Experimental Analysis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0102658

Computational docking models of ligands in the ER binding pocket. The protein backbone is shown as grey cartoon; ligands and side chain of residue 421 are shown as sticks; hydrogen atoms are not shown for clarity. M421F-ER α LBD mutant bound to bisphenol-A (orange in B), 4-nonylphenol (green in C) and 17β-estradiol (red in D). In contrast to the wt-ER α LBD (shown in A with bisphenol A), the aromatic ring of the F421 mutated ER may increase the affinity for the ligands either by direct stacking interactions or by increasing the aromatic content of the ligand binding pocket; no steric clashes are created by the incorporation of this larger side chain.
Figure Legend Snippet: Computational docking models of ligands in the ER binding pocket. The protein backbone is shown as grey cartoon; ligands and side chain of residue 421 are shown as sticks; hydrogen atoms are not shown for clarity. M421F-ER α LBD mutant bound to bisphenol-A (orange in B), 4-nonylphenol (green in C) and 17β-estradiol (red in D). In contrast to the wt-ER α LBD (shown in A with bisphenol A), the aromatic ring of the F421 mutated ER may increase the affinity for the ligands either by direct stacking interactions or by increasing the aromatic content of the ligand binding pocket; no steric clashes are created by the incorporation of this larger side chain.

Techniques Used: Binding Assay, Mutagenesis, Ligand Binding Assay

Competitive binding assay. Competitive binding assay on wt-ER α LBD (blue squares), M421F-ER α LBD (red circles) and M421I-ER α LBD (green triangles) with six different compounds: 17β-estradiol (panel A), 17α-ethinylestradiol (panel B), bisphenol-A (panel C), tamoxifen (panel D), 4-nonylphenol (panel E) and 4-tert-octylphenol (panel F).
Figure Legend Snippet: Competitive binding assay. Competitive binding assay on wt-ER α LBD (blue squares), M421F-ER α LBD (red circles) and M421I-ER α LBD (green triangles) with six different compounds: 17β-estradiol (panel A), 17α-ethinylestradiol (panel B), bisphenol-A (panel C), tamoxifen (panel D), 4-nonylphenol (panel E) and 4-tert-octylphenol (panel F).

Techniques Used: Competitive Binding Assay

31) Product Images from "Rational Modification of Estrogen Receptor by Combination of Computational and Experimental Analysis"

Article Title: Rational Modification of Estrogen Receptor by Combination of Computational and Experimental Analysis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0102658

Competitive binding assay. Competitive binding assay on wt-ER α LBD (blue squares), M421F-ER α LBD (red circles) and M421I-ER α LBD (green triangles) with six different compounds: 17β-estradiol (panel A), 17α-ethinylestradiol (panel B), bisphenol-A (panel C), tamoxifen (panel D), 4-nonylphenol (panel E) and 4-tert-octylphenol (panel F).
Figure Legend Snippet: Competitive binding assay. Competitive binding assay on wt-ER α LBD (blue squares), M421F-ER α LBD (red circles) and M421I-ER α LBD (green triangles) with six different compounds: 17β-estradiol (panel A), 17α-ethinylestradiol (panel B), bisphenol-A (panel C), tamoxifen (panel D), 4-nonylphenol (panel E) and 4-tert-octylphenol (panel F).

Techniques Used: Competitive Binding Assay

32) Product Images from "Soluble ST2 suppresses the effect of interleukin-33 on lung type 2 innate lymphoid cells"

Article Title: Soluble ST2 suppresses the effect of interleukin-33 on lung type 2 innate lymphoid cells

Journal: Biochemistry and Biophysics Reports

doi: 10.1016/j.bbrep.2016.02.002

The effect of soluble ST2 on the IL-33 signaling pathway in EL-4 cells that stably express ST2L. (A) Purified ST2-V5 (100 ng) was left untreated or treated with N -glycosidase F and developed on SDS polyacrylamide gel, followed by silver staining. (B) ST2L/EL-4 cells were pretreated with ST2-V5 or In-ST2-V5 (4.1, 20.7, and 103.6 ng/ml, corresponding to 0.11, 0.56, and 2.80 nM, respectively) for 1 h and then stimulated with IL-33 (10 ng/ml, corresponding to 0.56 nM) for 15 min. PBS was added as a control for ST2-V5, In-ST2-V5, and IL-33. Phosphorylation and expression levels of the indicated proteins were detected by western blotting. Results are representative of three independent experiments. p-, phosphorylated.
Figure Legend Snippet: The effect of soluble ST2 on the IL-33 signaling pathway in EL-4 cells that stably express ST2L. (A) Purified ST2-V5 (100 ng) was left untreated or treated with N -glycosidase F and developed on SDS polyacrylamide gel, followed by silver staining. (B) ST2L/EL-4 cells were pretreated with ST2-V5 or In-ST2-V5 (4.1, 20.7, and 103.6 ng/ml, corresponding to 0.11, 0.56, and 2.80 nM, respectively) for 1 h and then stimulated with IL-33 (10 ng/ml, corresponding to 0.56 nM) for 15 min. PBS was added as a control for ST2-V5, In-ST2-V5, and IL-33. Phosphorylation and expression levels of the indicated proteins were detected by western blotting. Results are representative of three independent experiments. p-, phosphorylated.

Techniques Used: Stable Transfection, Purification, Silver Staining, Expressing, Western Blot

33) Product Images from "Existing Antilisterial Immunity Does Not Inhibit the Development of a Listeria monocytogenes-Specific Primary Cytotoxic T-Lymphocyte Response"

Article Title: Existing Antilisterial Immunity Does Not Inhibit the Development of a Listeria monocytogenes-Specific Primary Cytotoxic T-Lymphocyte Response

Journal: Infection and Immunity

doi:

p60 217–225-specific CTLs are absent from mice immunized with the p60 218F mutant. BALB/c mice were immunized either with wild-type L. monocytogenes , the 92F mutant, or the p60 218F mutant. Six days later, spleen cells were stimulated in culture for 72 h, and the recovered cells were utilized as effector CTLs. 51 CR-labeled RMAS-K d cells were pulsed with a 10 −9 M concentration of either the LLO 91–99 (solid bars) or p60 217–225 (stippled bars) peptide for 60 min and then added at 10 4 cells/well. Effector CTLs were added at an E/T ratio of 50:1. The assay was terminated 4 h later, and the percent 51 Cr was determined. The data are representative of four experiments.
Figure Legend Snippet: p60 217–225-specific CTLs are absent from mice immunized with the p60 218F mutant. BALB/c mice were immunized either with wild-type L. monocytogenes , the 92F mutant, or the p60 218F mutant. Six days later, spleen cells were stimulated in culture for 72 h, and the recovered cells were utilized as effector CTLs. 51 CR-labeled RMAS-K d cells were pulsed with a 10 −9 M concentration of either the LLO 91–99 (solid bars) or p60 217–225 (stippled bars) peptide for 60 min and then added at 10 4 cells/well. Effector CTLs were added at an E/T ratio of 50:1. The assay was terminated 4 h later, and the percent 51 Cr was determined. The data are representative of four experiments.

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

Effector CTLs from mice immunized with the p60 218F mutant lyse L. monocytogenes -infected target cells. BALB/c mice were immunized either with wild-type L. monocytogenes , the 92F mutant or the p60 218F mutant. Six days later, spleen cells were stimulated in culture for 72 h, and the recovered cells were utilized as effector CTLs. J774 target cells were infected with wild-type L. monocytogenes at an MOI of 2:1 to 5:1 and washed 60 min later, and 40 μg of gentamicin per ml was added. Three hours later, effector CTLs at an E/T ratio of 20:1 (solid bars) or 5:1 (stippled bars) were added. The assay was terminated 4 h later, and the percent CFU reduction was determined. The data are representative of four experiments. The mean number of CFU per well for L. monocytogenes -infected J774 cells in the absence of antilisterial CTLs for this experiment was 9.2 × 10 6 .
Figure Legend Snippet: Effector CTLs from mice immunized with the p60 218F mutant lyse L. monocytogenes -infected target cells. BALB/c mice were immunized either with wild-type L. monocytogenes , the 92F mutant or the p60 218F mutant. Six days later, spleen cells were stimulated in culture for 72 h, and the recovered cells were utilized as effector CTLs. J774 target cells were infected with wild-type L. monocytogenes at an MOI of 2:1 to 5:1 and washed 60 min later, and 40 μg of gentamicin per ml was added. Three hours later, effector CTLs at an E/T ratio of 20:1 (solid bars) or 5:1 (stippled bars) were added. The assay was terminated 4 h later, and the percent CFU reduction was determined. The data are representative of four experiments. The mean number of CFU per well for L. monocytogenes -infected J774 cells in the absence of antilisterial CTLs for this experiment was 9.2 × 10 6 .

Techniques Used: Mouse Assay, Mutagenesis, Infection

34) Product Images from "Mesenchymal stem cells derived from human iPS cells via mesoderm and neuroepithelium have different features and therapeutic potentials"

Article Title: Mesenchymal stem cells derived from human iPS cells via mesoderm and neuroepithelium have different features and therapeutic potentials

Journal: PLoS ONE

doi: 10.1371/journal.pone.0200790

DNA microarray analysis of PSP-MSCs and RA-Pα-MSCs. (A-C): Hierarchical clustering of gene sets signatures, pluripotent markers (A), MSC markers (B) and paracrine factors (C), in various MSCs and N1-12 iPSCs. The datasets of all genes investigated were clustered according to Euclidean distance metrics. The labels represent the following cells: N1-12; N1-12 iPS cells, BM-MSC; BM-MSC (PRC-010) purchased from Bay bioscience Co.; N1-12 PSP; PSP-MSC derived from N1-12, 201B7 PSP; PSP-MSC derived from 201B7, N1-12 RA-Pα; RA-Pα-MSC derived from N1-12, 201B7 RA-Pα; RA-Pa-MSC derived from 201B7. (D): Principal component analysis. All datasets were classified into three principal components, PC2 (25.43%), PC3 (15.34%), and PC4 (7.81%), and were simplified into three-dimensional scores. (E, F): Gene ontology (GO) analysis of 286 commonly upregulated data sets for PSP-MSC (E) and 359 data sets for RA-Pα-MSC (F). The top-ten GO terms are listed. GO terms were detected with a cutoff P-value of 0.1. Values are–log10 corrected P-value. Red color indicates different GO terms between (E) and (F).
Figure Legend Snippet: DNA microarray analysis of PSP-MSCs and RA-Pα-MSCs. (A-C): Hierarchical clustering of gene sets signatures, pluripotent markers (A), MSC markers (B) and paracrine factors (C), in various MSCs and N1-12 iPSCs. The datasets of all genes investigated were clustered according to Euclidean distance metrics. The labels represent the following cells: N1-12; N1-12 iPS cells, BM-MSC; BM-MSC (PRC-010) purchased from Bay bioscience Co.; N1-12 PSP; PSP-MSC derived from N1-12, 201B7 PSP; PSP-MSC derived from 201B7, N1-12 RA-Pα; RA-Pα-MSC derived from N1-12, 201B7 RA-Pα; RA-Pa-MSC derived from 201B7. (D): Principal component analysis. All datasets were classified into three principal components, PC2 (25.43%), PC3 (15.34%), and PC4 (7.81%), and were simplified into three-dimensional scores. (E, F): Gene ontology (GO) analysis of 286 commonly upregulated data sets for PSP-MSC (E) and 359 data sets for RA-Pα-MSC (F). The top-ten GO terms are listed. GO terms were detected with a cutoff P-value of 0.1. Values are–log10 corrected P-value. Red color indicates different GO terms between (E) and (F).

Techniques Used: Microarray, Derivative Assay

Induction of hiPSC-derived MSCs under mesodermal and neuroepithelial differentiation conditions. (A, B): The proportions of PDGFRα and VEGFR2 expression in differentiated N1-12 cells on day 2, 4, 6 and 8 under the mesodermal differentiation condition. Representative data (A) and graph (B). Number indicates the percentage of each population (A). Experiments were conducted three times (mean ± SD). (C): Quantitative PCR (qPCR) analysis of the relative mRNA levels of NANOG and OCT3/4 as multipotent markers, VIMENTIN and PDGFRβ for mesenchyme, BRACHYURY for mesoderm, and SOX1 for neuroepithelium during the mesodermal (left) and neuroepithelial (right) differentiations. Symbols: d-number indicates day-number of differentiation, PSP; immediately after sorting on day 6 under the mesoderm differentiation, RA-Pα: immediately after sorting on day 10 under the neuroepithelial differentiation, P3 or P6; passage 3 or 6 after sorting. Each value represents the mean fold compared to day-0 undifferentiated iPSCs. Each experiment was conducted three times (mean ± SD). (D): Bright-light image of PSP-MSC and RA-Pα cells on day 21 after sorting. Scale bar: 200 μm. (E): The proportion of PDGFRα and VEGFR2 expressions in day-10 differentiated N1-12 under the neuroepithelial differentiation condition; number indicates the percentage of each population.
Figure Legend Snippet: Induction of hiPSC-derived MSCs under mesodermal and neuroepithelial differentiation conditions. (A, B): The proportions of PDGFRα and VEGFR2 expression in differentiated N1-12 cells on day 2, 4, 6 and 8 under the mesodermal differentiation condition. Representative data (A) and graph (B). Number indicates the percentage of each population (A). Experiments were conducted three times (mean ± SD). (C): Quantitative PCR (qPCR) analysis of the relative mRNA levels of NANOG and OCT3/4 as multipotent markers, VIMENTIN and PDGFRβ for mesenchyme, BRACHYURY for mesoderm, and SOX1 for neuroepithelium during the mesodermal (left) and neuroepithelial (right) differentiations. Symbols: d-number indicates day-number of differentiation, PSP; immediately after sorting on day 6 under the mesoderm differentiation, RA-Pα: immediately after sorting on day 10 under the neuroepithelial differentiation, P3 or P6; passage 3 or 6 after sorting. Each value represents the mean fold compared to day-0 undifferentiated iPSCs. Each experiment was conducted three times (mean ± SD). (D): Bright-light image of PSP-MSC and RA-Pα cells on day 21 after sorting. Scale bar: 200 μm. (E): The proportion of PDGFRα and VEGFR2 expressions in day-10 differentiated N1-12 under the neuroepithelial differentiation condition; number indicates the percentage of each population.

Techniques Used: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction

35) Product Images from "Alkylation of Galectin-1 with Iodoacetamide and Mass Spectrometric Mapping of the Sites of Incorporation"

Article Title: Alkylation of Galectin-1 with Iodoacetamide and Mass Spectrometric Mapping of the Sites of Incorporation

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-4939-1396-1_3

Alkylation protects Gal-1 from oxidative inactivation. Galectin-1 (Gal-1) or iodoacetamide-treated Gal-1 (iGal-1) was incubated for 24 h in PBS at 37 °C followed by subjection to affinity chromatography over lactosyl-Sepharose. This research was originally published in the Journal of Biological Chemistry. Stowell SR, Cho M, Feasley CL, Arthur CM, Song X, Colucci JK, Karmakar S, Mehta P, Dias-Baruffi M, McEver RP, Cummings RD. Ligand reduces galectin-1 sensitivity to oxidative inactivation by enhancing dimer formation. 2009 Feb 20;284(8):4989-99 © the American Society for Biochemistry and Molecular Biology
Figure Legend Snippet: Alkylation protects Gal-1 from oxidative inactivation. Galectin-1 (Gal-1) or iodoacetamide-treated Gal-1 (iGal-1) was incubated for 24 h in PBS at 37 °C followed by subjection to affinity chromatography over lactosyl-Sepharose. This research was originally published in the Journal of Biological Chemistry. Stowell SR, Cho M, Feasley CL, Arthur CM, Song X, Colucci JK, Karmakar S, Mehta P, Dias-Baruffi M, McEver RP, Cummings RD. Ligand reduces galectin-1 sensitivity to oxidative inactivation by enhancing dimer formation. 2009 Feb 20;284(8):4989-99 © the American Society for Biochemistry and Molecular Biology

Techniques Used: Incubation, Affinity Chromatography

36) Product Images from "An Electrochemical Fiveplex Biochip Assay Based on Anti-Idiotypic Antibodies for Fast On-Site Detection of Bioterrorism Relevant Low Molecular Weight Toxins"

Article Title: An Electrochemical Fiveplex Biochip Assay Based on Anti-Idiotypic Antibodies for Fast On-Site Detection of Bioterrorism Relevant Low Molecular Weight Toxins

Journal: Toxins

doi: 10.3390/toxins11120696

Schematic procedure of the automated fiveplex anti-idiotypic antibody-based competitive immunoassay for detection of STX, MC-LR, T-2, RoA and AFB1 upon an electrochemical biochip. ( 1 ) Application of the sample mixed with monoclonal detection antibody-β-D-galactosidase (mAb-bGAL) tracer cocktail to the biochip pre-coated with capture mAbs; ( 2 ) Competition reaction upon the biochip; ( 3 ) bGAL substrate flow as well as amperometric readout of each electrode position. (Note: Illustrated biochip scheme represents only a small section of the entire electrode positions).
Figure Legend Snippet: Schematic procedure of the automated fiveplex anti-idiotypic antibody-based competitive immunoassay for detection of STX, MC-LR, T-2, RoA and AFB1 upon an electrochemical biochip. ( 1 ) Application of the sample mixed with monoclonal detection antibody-β-D-galactosidase (mAb-bGAL) tracer cocktail to the biochip pre-coated with capture mAbs; ( 2 ) Competition reaction upon the biochip; ( 3 ) bGAL substrate flow as well as amperometric readout of each electrode position. (Note: Illustrated biochip scheme represents only a small section of the entire electrode positions).

Techniques Used: Flow Cytometry

37) Product Images from "The structure of the nucleoprotein of Influenza D shows that all Orthomyxoviridae nucleoproteins have a similar NPCORE, with or without a NPTAIL for nuclear transport"

Article Title: The structure of the nucleoprotein of Influenza D shows that all Orthomyxoviridae nucleoproteins have a similar NPCORE, with or without a NPTAIL for nuclear transport

Journal: Scientific Reports

doi: 10.1038/s41598-018-37306-y

Interaction of D/NP and D/NP TAIL with importin-α7. ( a ) Size exclusion chromatography profile of a mixture between human importin-α7 and D/NP TAIL . The mixture (molar ratio 1 importin-α7 for 2 D/NP TAIL ) was incubated 1 hour at room temperature and then loaded on a Superdex TM 75 10/300GL column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM β-mercaptoethanol. ( b ) Thermal stability assay of importin-α7 in absence (green) or in presence (red) of D/NP TAIL using Thermofluor 76 . In presence of D/NP TAIL , the melting temperature of importin-α7 is 5 °C higher. D/NP TAIL alone using Thermofluor did not give any denaturation signal (yellow curve). The upper insert corresponds to the derivative of the fluorescence signal for a precise measure of the melting temperature. ( c ) Affinity of importin-α7 for D/NP TAIL by measured by surface plasmon resonance (SPR). Biotinylated D/NP TAIL (left) and control peptide (right) were captured on a streptavidin-coated sensor chip surface before injections of several importin-α7 concentrations (10 nM in red, 25 nM in orange, 50 nM in green, 75 nM in blue and 100 nM in purple). The sensorgrams of the interaction between D/NP TAIL and importin-α7 were fitted under a Langmuir 1:1 binding model with mass-transfer (black line). ( d ) SEC-MALLS analysis of D/NP in complex with importin-α7. The mixture (molar ratio 1 D/NP for 1.2 importin-α7) was incubated 1 hour at room temperature and then loaded on a Superdex TM 200 increase 10/300 GL. The experimental molecular weight is consistent with the expected mass of four importins-α7 bound per D/NP tetramer. ( e ) Pull-down assays of human importin-α7 by D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511). The his-tags are on D/NP. The mixtures (molar ratio 1 D/NP for 1.2 importin-α7) were incubated 1 hour and the experience was done as described in panel ( a ). The figure shows the coomassie blue-stained SDS-PAGE (12% polyacrylamide) with the Load, FlowThough, Wash and the second fractions (E2).
Figure Legend Snippet: Interaction of D/NP and D/NP TAIL with importin-α7. ( a ) Size exclusion chromatography profile of a mixture between human importin-α7 and D/NP TAIL . The mixture (molar ratio 1 importin-α7 for 2 D/NP TAIL ) was incubated 1 hour at room temperature and then loaded on a Superdex TM 75 10/300GL column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM β-mercaptoethanol. ( b ) Thermal stability assay of importin-α7 in absence (green) or in presence (red) of D/NP TAIL using Thermofluor 76 . In presence of D/NP TAIL , the melting temperature of importin-α7 is 5 °C higher. D/NP TAIL alone using Thermofluor did not give any denaturation signal (yellow curve). The upper insert corresponds to the derivative of the fluorescence signal for a precise measure of the melting temperature. ( c ) Affinity of importin-α7 for D/NP TAIL by measured by surface plasmon resonance (SPR). Biotinylated D/NP TAIL (left) and control peptide (right) were captured on a streptavidin-coated sensor chip surface before injections of several importin-α7 concentrations (10 nM in red, 25 nM in orange, 50 nM in green, 75 nM in blue and 100 nM in purple). The sensorgrams of the interaction between D/NP TAIL and importin-α7 were fitted under a Langmuir 1:1 binding model with mass-transfer (black line). ( d ) SEC-MALLS analysis of D/NP in complex with importin-α7. The mixture (molar ratio 1 D/NP for 1.2 importin-α7) was incubated 1 hour at room temperature and then loaded on a Superdex TM 200 increase 10/300 GL. The experimental molecular weight is consistent with the expected mass of four importins-α7 bound per D/NP tetramer. ( e ) Pull-down assays of human importin-α7 by D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511). The his-tags are on D/NP. The mixtures (molar ratio 1 D/NP for 1.2 importin-α7) were incubated 1 hour and the experience was done as described in panel ( a ). The figure shows the coomassie blue-stained SDS-PAGE (12% polyacrylamide) with the Load, FlowThough, Wash and the second fractions (E2).

Techniques Used: Size-exclusion Chromatography, Incubation, Stability Assay, Fluorescence, SPR Assay, Chromatin Immunoprecipitation, Binding Assay, Molecular Weight, Staining, SDS Page

Purification and characterized of Influenza D nucleoprotein. ( a ) Size exclusion chromatography profile of wild-type D/NP. The sample was loaded on a Hiload TM 16/600 S200 column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 300 mM NaCl and 5 mM β-mercaptoethanol. ( b ) SEC-MALLS-RI analysis of D/NP. SEC was performed with a Superdex TM 200 increase 10/300 GL column equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5 mM β-ME. The panel shows the theoretical Mw and the measured Mw. ( c ) and ( e ) Electron microscopy images of the elution peak of D/NP and D/NP-511. Samples show different oligomeric states although most oligomers are tetramers. The scale bar corresponds to 100 nm. ( d ) Coomassie blue-stained SDS-PAGE (4–20% gradient polyacrylamide) showing the purified wild-type D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511).
Figure Legend Snippet: Purification and characterized of Influenza D nucleoprotein. ( a ) Size exclusion chromatography profile of wild-type D/NP. The sample was loaded on a Hiload TM 16/600 S200 column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 300 mM NaCl and 5 mM β-mercaptoethanol. ( b ) SEC-MALLS-RI analysis of D/NP. SEC was performed with a Superdex TM 200 increase 10/300 GL column equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5 mM β-ME. The panel shows the theoretical Mw and the measured Mw. ( c ) and ( e ) Electron microscopy images of the elution peak of D/NP and D/NP-511. Samples show different oligomeric states although most oligomers are tetramers. The scale bar corresponds to 100 nm. ( d ) Coomassie blue-stained SDS-PAGE (4–20% gradient polyacrylamide) showing the purified wild-type D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511).

Techniques Used: Purification, Size-exclusion Chromatography, Electron Microscopy, Staining, SDS Page

38) Product Images from "The Signaling Network of Transforming Growth Factor ?1, Protein Kinase C?, and Integrin Underlies the Spreading and Invasiveness of Gastric Carcinoma Cells"

Article Title: The Signaling Network of Transforming Growth Factor ?1, Protein Kinase C?, and Integrin Underlies the Spreading and Invasiveness of Gastric Carcinoma Cells

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.25.16.6921-6936.2005

The TGFβ1 effects depend on expression and activation of integrins α2 or α3. The cells were pretreated with 10 μg/ml anti-integrin α2 (P1E6), α3 (P1B5), or α5 (P1D6) antibodies, 30 min prior to the replating on Fn and a concomitant TGFβ1 treatment for 20 h. After the incubation, cell images were taken, and lysates were prepared and used in immunoblots using antibodies against the indicated molecules. Data shown are representative of two isolated experiments. (A) Functional blocking anti-integrin α2 or α3, but not α5, antibodies abolished the cell spreading. (B) Functional blocking of the integrins inhibited phosphorylation of the FA molecules. (C) Cells were transiently cotransfected with a control plasmid (C) or pSF2-integrin α2 or α3 with pCDNA3-GFP. One day after the transfection, cells were replated onto Fn (10 μg/ml)-precoated cover glasses in the absence of TGFβ1 treatment. After incubation for 20 h at 37°C and 5% CO 2 , cells were processed for actin staining with phalloidin-conjugated TRITC. Another set of cells in 60-mm culture dishes were harvested for lysates prior to performing immunoblotting using the indicated antibodies. BLK mAb, functional blocking monoclonal antibody.
Figure Legend Snippet: The TGFβ1 effects depend on expression and activation of integrins α2 or α3. The cells were pretreated with 10 μg/ml anti-integrin α2 (P1E6), α3 (P1B5), or α5 (P1D6) antibodies, 30 min prior to the replating on Fn and a concomitant TGFβ1 treatment for 20 h. After the incubation, cell images were taken, and lysates were prepared and used in immunoblots using antibodies against the indicated molecules. Data shown are representative of two isolated experiments. (A) Functional blocking anti-integrin α2 or α3, but not α5, antibodies abolished the cell spreading. (B) Functional blocking of the integrins inhibited phosphorylation of the FA molecules. (C) Cells were transiently cotransfected with a control plasmid (C) or pSF2-integrin α2 or α3 with pCDNA3-GFP. One day after the transfection, cells were replated onto Fn (10 μg/ml)-precoated cover glasses in the absence of TGFβ1 treatment. After incubation for 20 h at 37°C and 5% CO 2 , cells were processed for actin staining with phalloidin-conjugated TRITC. Another set of cells in 60-mm culture dishes were harvested for lysates prior to performing immunoblotting using the indicated antibodies. BLK mAb, functional blocking monoclonal antibody.

Techniques Used: Expressing, Activation Assay, Incubation, Western Blot, Isolation, Functional Assay, Blocking Assay, Plasmid Preparation, Transfection, Staining

Schematic working model of the TGFβ1-, PKCδ-, and integrin-mediated cellular functions in gastric carcinoma cells. TGFβ1 treatments of cells on ECMs lead to increased expression and phosphorylation of PKCδ, cell surface levels of integrins α2 or α3, and activation of the focal adhesion molecules. This signaling network results in spreading, migration, and invasion of the SNU16mAd gastric carcinoma cell variant. In addition to a linear connection, presumably additional bypassing connections may contribute to the TGFβ1 effects.
Figure Legend Snippet: Schematic working model of the TGFβ1-, PKCδ-, and integrin-mediated cellular functions in gastric carcinoma cells. TGFβ1 treatments of cells on ECMs lead to increased expression and phosphorylation of PKCδ, cell surface levels of integrins α2 or α3, and activation of the focal adhesion molecules. This signaling network results in spreading, migration, and invasion of the SNU16mAd gastric carcinoma cell variant. In addition to a linear connection, presumably additional bypassing connections may contribute to the TGFβ1 effects.

Techniques Used: Expressing, Activation Assay, Migration, Variant Assay

39) Product Images from "Regulation of Multiple Cytokine Signalling Pathways by SOCS3 is Independent of SOCS2"

Article Title: Regulation of Multiple Cytokine Signalling Pathways by SOCS3 is Independent of SOCS2

Journal:

doi: 10.3109/08977190903210954

SOCS3 expression, STAT5 phosphorylation and IL-2 induced proliferation are unaltered in Socs2 −/− lymphocytes stimulated with IL-2. (A) WT, Socs2 −/− and Socs3 −/ Δ vav LN cells were stimulated with mIL-2 (10
Figure Legend Snippet: SOCS3 expression, STAT5 phosphorylation and IL-2 induced proliferation are unaltered in Socs2 −/− lymphocytes stimulated with IL-2. (A) WT, Socs2 −/− and Socs3 −/ Δ vav LN cells were stimulated with mIL-2 (10

Techniques Used: Expressing

40) Product Images from "Structural basis of chimpanzee APOBEC3H dimerization stabilized by double-stranded RNA"

Article Title: Structural basis of chimpanzee APOBEC3H dimerization stabilized by double-stranded RNA

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky676

Effect of cpzA3H mutations in the RNA recognition interface on the intracellular levels of cpzA3H. WT cpzA3H and mutants were transiently expressed in 293T cells in the absence of an inhibitor or in the presence of MG132 (8 μM), chloroquine (100 μM) or bafilomycin A1 (80 nM). The proteins were detected by western blotting with the anti-DYKDDDDK tag monoclonal antibody (Anti-FLAG) or anti-β-tubulin antibody (Anti-β-Tubulin).
Figure Legend Snippet: Effect of cpzA3H mutations in the RNA recognition interface on the intracellular levels of cpzA3H. WT cpzA3H and mutants were transiently expressed in 293T cells in the absence of an inhibitor or in the presence of MG132 (8 μM), chloroquine (100 μM) or bafilomycin A1 (80 nM). The proteins were detected by western blotting with the anti-DYKDDDDK tag monoclonal antibody (Anti-FLAG) or anti-β-tubulin antibody (Anti-β-Tubulin).

Techniques Used: Western Blot

Related Articles

Transduction:

Article Title: Mutated NPM1 in combination with overexpression of Meis1 or Hoxa9 is not sufficient to induce acute myeloid leukemia
Article Snippet: .. In brief, murine BM cells harvested from C57BL/6 J mice 4 days after 150 µg/g 5-fluorouracil (5-FU, Accord Healthcare AB, Solna, Sweden) treatment were transduced with NPMc+ and neo or YFP viruses (NPMc+ cells), Meis1 and GFP viruses (Meis1 cells), Hoxa9 and GFP viruses (Hoxa9 cells), Meis1 and NPMc+ viruses (Meis1-NPMc+ cells) or Hoxa9 and NPMc+ viruses (Hoxa9-NPMc+ cells) and cultured in Dulbecco modified Eagle medium (DMEM with high glucose, D6429, Sigma-Aldrich Sweden AB, Stockholm, Sweden) supplemented with 15 % fetal bovine serum (6250, StemCell Technologies Inc., Vancouver, Canada), 2 mM l -glutamine (G7513-100 ml, Sigma-Aldrich Sweden AB, Stockholm, Sweden), 1 % Penicillin and streptomycin (P4333-100 ml, Sigma-Aldrich Sweden AB, Stockholm, Sweden), 10 ng/ml human interleukin-6 (2506, StemCell Technologies SARL, Grenoble, France), 6 ng/ml murine interleukin-3 (2733, StemCell Technologies SARL, Grenoble, France), and 50 ng/ml murine stem cell factor (2931 StemCell Technologies SARL, Grenoble, France) (complete medium). .. Transduction was achieved by co-culturing 5-FU treated murine BM cells with irradiated (2 × 25 Gray) GP + E86 viral cells in complete medium supplemented with 5 µg/ml protamine sulfate (P-4020, Sigma-Aldrich Sweden AB, Stockholm, Sweden).

Article Title: Role of donor and host cells in muscle-derived stem cell-mediated bone repair: differentiation vs. paracrine effects
Article Snippet: .. MDSCs were transduced with retro-GFP or retro-BMP4/GFP at a multiplicity of infection of 5 in the presence of 8 μg/ml polybrene (Sigma-Aldrich, Milwaukee, WI, USA). .. The cells were then expanded for 4 passages, after which the GFP+ cells were selected by fluorescence-activated cell sorting (FACS; BD FACSAria IIu; BD Biosciences, Bedford, MA, USA).

Immunohistochemistry:

Article Title: The Effect of Transient Local Anti-inflammatory Treatment on the Survival of Pig Retinal Progenitor Cell Allotransplants
Article Snippet: .. Dual-label IHC was performed using a cocktail of anti-GFP (Abcam, ab13970 1:500) to detect GFP pRPCs and anti-Ki-67 (Vector, VP-K452 1:100; proliferation), anti-recoverin (Millipore, AB5585 1:500; cones), or mouse anti-rhodopsin (Millipore, MABN15 1:500; rods) antibodies. .. For infiltrating leukocytes/macrophages, we used anti-CD45 antibody (Abcam, 1:200).

Mouse Assay:

Article Title: Mutated NPM1 in combination with overexpression of Meis1 or Hoxa9 is not sufficient to induce acute myeloid leukemia
Article Snippet: .. In brief, murine BM cells harvested from C57BL/6 J mice 4 days after 150 µg/g 5-fluorouracil (5-FU, Accord Healthcare AB, Solna, Sweden) treatment were transduced with NPMc+ and neo or YFP viruses (NPMc+ cells), Meis1 and GFP viruses (Meis1 cells), Hoxa9 and GFP viruses (Hoxa9 cells), Meis1 and NPMc+ viruses (Meis1-NPMc+ cells) or Hoxa9 and NPMc+ viruses (Hoxa9-NPMc+ cells) and cultured in Dulbecco modified Eagle medium (DMEM with high glucose, D6429, Sigma-Aldrich Sweden AB, Stockholm, Sweden) supplemented with 15 % fetal bovine serum (6250, StemCell Technologies Inc., Vancouver, Canada), 2 mM l -glutamine (G7513-100 ml, Sigma-Aldrich Sweden AB, Stockholm, Sweden), 1 % Penicillin and streptomycin (P4333-100 ml, Sigma-Aldrich Sweden AB, Stockholm, Sweden), 10 ng/ml human interleukin-6 (2506, StemCell Technologies SARL, Grenoble, France), 6 ng/ml murine interleukin-3 (2733, StemCell Technologies SARL, Grenoble, France), and 50 ng/ml murine stem cell factor (2931 StemCell Technologies SARL, Grenoble, France) (complete medium). .. Transduction was achieved by co-culturing 5-FU treated murine BM cells with irradiated (2 × 25 Gray) GP + E86 viral cells in complete medium supplemented with 5 µg/ml protamine sulfate (P-4020, Sigma-Aldrich Sweden AB, Stockholm, Sweden).

Immunolabeling:

Article Title: AFAP1L1 is a novel adaptor protein of the AFAP family that interacts with cortactin and localizes to invadosomes
Article Snippet: .. Supplemental Figure 2: Colocalization of GFP-AFAP1L1 and cortactin: A7r5 cells transiently expressing GFP-AFAP1L1 were plated onto fibronectin-coated coverslips and immunolabeled for cortactin (Millipore). ..

Cell Culture:

Article Title: Mutated NPM1 in combination with overexpression of Meis1 or Hoxa9 is not sufficient to induce acute myeloid leukemia
Article Snippet: .. In brief, murine BM cells harvested from C57BL/6 J mice 4 days after 150 µg/g 5-fluorouracil (5-FU, Accord Healthcare AB, Solna, Sweden) treatment were transduced with NPMc+ and neo or YFP viruses (NPMc+ cells), Meis1 and GFP viruses (Meis1 cells), Hoxa9 and GFP viruses (Hoxa9 cells), Meis1 and NPMc+ viruses (Meis1-NPMc+ cells) or Hoxa9 and NPMc+ viruses (Hoxa9-NPMc+ cells) and cultured in Dulbecco modified Eagle medium (DMEM with high glucose, D6429, Sigma-Aldrich Sweden AB, Stockholm, Sweden) supplemented with 15 % fetal bovine serum (6250, StemCell Technologies Inc., Vancouver, Canada), 2 mM l -glutamine (G7513-100 ml, Sigma-Aldrich Sweden AB, Stockholm, Sweden), 1 % Penicillin and streptomycin (P4333-100 ml, Sigma-Aldrich Sweden AB, Stockholm, Sweden), 10 ng/ml human interleukin-6 (2506, StemCell Technologies SARL, Grenoble, France), 6 ng/ml murine interleukin-3 (2733, StemCell Technologies SARL, Grenoble, France), and 50 ng/ml murine stem cell factor (2931 StemCell Technologies SARL, Grenoble, France) (complete medium). .. Transduction was achieved by co-culturing 5-FU treated murine BM cells with irradiated (2 × 25 Gray) GP + E86 viral cells in complete medium supplemented with 5 µg/ml protamine sulfate (P-4020, Sigma-Aldrich Sweden AB, Stockholm, Sweden).

Size-exclusion Chromatography:

Article Title: N-glycosylation converts non-glycoproteins into mannose receptor ligands and reveals antigen-specific T cell responses in vivo
Article Snippet: .. For purification of NST-GFP, NST-GFP-S8L and QST-GFP, proteins were additionally purified by size exclusion chromatography (Sephacryl S200 HR, Sigma, Taufkirchen, Germany). .. Expression and purification of NST-GFP and QST-GFP from HEK293T cells NST-GFP and QST-GFP were cloned behind the IL-2 secretion sequence of pIgFuse, thereby removing the IgG sequence, and transfected into HEK293T cells.

Incubation:

Article Title: Drosophila cyclin D/Cdk4 regulates mitochondrial biogenesis and aging and sensitizes animals to hypoxic stress
Article Snippet: .. 96AED En-Gal4 , UAS-GFP/UAS-X animals were dissected in PBS, incubated at RT with stained with 30 µM DHE (Sigma Aldrich D7008) solution for 10–15 min. ..

Infection:

Article Title: Role of donor and host cells in muscle-derived stem cell-mediated bone repair: differentiation vs. paracrine effects
Article Snippet: .. MDSCs were transduced with retro-GFP or retro-BMP4/GFP at a multiplicity of infection of 5 in the presence of 8 μg/ml polybrene (Sigma-Aldrich, Milwaukee, WI, USA). .. The cells were then expanded for 4 passages, after which the GFP+ cells were selected by fluorescence-activated cell sorting (FACS; BD FACSAria IIu; BD Biosciences, Bedford, MA, USA).

Expressing:

Article Title: AFAP1L1 is a novel adaptor protein of the AFAP family that interacts with cortactin and localizes to invadosomes
Article Snippet: .. Supplemental Figure 2: Colocalization of GFP-AFAP1L1 and cortactin: A7r5 cells transiently expressing GFP-AFAP1L1 were plated onto fibronectin-coated coverslips and immunolabeled for cortactin (Millipore). ..

Article Title: Simple rules for passive diffusion through the nuclear pore complex
Article Snippet: .. These GFP-xPrA genes were subcloned either into the yeast constitutive expression plasmid pYX242 (EMD Millipore, discontinued) or as 6xHIS fusion genes into the bacterial expression plasmid pET28a (EMD Millipore). .. GFP-1PrG and -2PrG constructs were made similarly, as yeast and bacterial expression constructs, by fusing to GFP either the C2 domain (for GFP-1PrG) or both the C2 and C3 domains (for GFP-2PrG) from Streptococcus protein G ( ).

Modification:

Article Title: Mutated NPM1 in combination with overexpression of Meis1 or Hoxa9 is not sufficient to induce acute myeloid leukemia
Article Snippet: .. In brief, murine BM cells harvested from C57BL/6 J mice 4 days after 150 µg/g 5-fluorouracil (5-FU, Accord Healthcare AB, Solna, Sweden) treatment were transduced with NPMc+ and neo or YFP viruses (NPMc+ cells), Meis1 and GFP viruses (Meis1 cells), Hoxa9 and GFP viruses (Hoxa9 cells), Meis1 and NPMc+ viruses (Meis1-NPMc+ cells) or Hoxa9 and NPMc+ viruses (Hoxa9-NPMc+ cells) and cultured in Dulbecco modified Eagle medium (DMEM with high glucose, D6429, Sigma-Aldrich Sweden AB, Stockholm, Sweden) supplemented with 15 % fetal bovine serum (6250, StemCell Technologies Inc., Vancouver, Canada), 2 mM l -glutamine (G7513-100 ml, Sigma-Aldrich Sweden AB, Stockholm, Sweden), 1 % Penicillin and streptomycin (P4333-100 ml, Sigma-Aldrich Sweden AB, Stockholm, Sweden), 10 ng/ml human interleukin-6 (2506, StemCell Technologies SARL, Grenoble, France), 6 ng/ml murine interleukin-3 (2733, StemCell Technologies SARL, Grenoble, France), and 50 ng/ml murine stem cell factor (2931 StemCell Technologies SARL, Grenoble, France) (complete medium). .. Transduction was achieved by co-culturing 5-FU treated murine BM cells with irradiated (2 × 25 Gray) GP + E86 viral cells in complete medium supplemented with 5 µg/ml protamine sulfate (P-4020, Sigma-Aldrich Sweden AB, Stockholm, Sweden).

Staining:

Article Title: Drosophila cyclin D/Cdk4 regulates mitochondrial biogenesis and aging and sensitizes animals to hypoxic stress
Article Snippet: .. 96AED En-Gal4 , UAS-GFP/UAS-X animals were dissected in PBS, incubated at RT with stained with 30 µM DHE (Sigma Aldrich D7008) solution for 10–15 min. ..

Purification:

Article Title: N-glycosylation converts non-glycoproteins into mannose receptor ligands and reveals antigen-specific T cell responses in vivo
Article Snippet: .. For purification of NST-GFP, NST-GFP-S8L and QST-GFP, proteins were additionally purified by size exclusion chromatography (Sephacryl S200 HR, Sigma, Taufkirchen, Germany). .. Expression and purification of NST-GFP and QST-GFP from HEK293T cells NST-GFP and QST-GFP were cloned behind the IL-2 secretion sequence of pIgFuse, thereby removing the IgG sequence, and transfected into HEK293T cells.

Plasmid Preparation:

Article Title: Simple rules for passive diffusion through the nuclear pore complex
Article Snippet: .. These GFP-xPrA genes were subcloned either into the yeast constitutive expression plasmid pYX242 (EMD Millipore, discontinued) or as 6xHIS fusion genes into the bacterial expression plasmid pET28a (EMD Millipore). .. GFP-1PrG and -2PrG constructs were made similarly, as yeast and bacterial expression constructs, by fusing to GFP either the C2 domain (for GFP-1PrG) or both the C2 and C3 domains (for GFP-2PrG) from Streptococcus protein G ( ).

Article Title: Venezuelan Equine Encephalitis Virus Capsid Protein Forms a Tetrameric Complex with CRM1 and Importin ?/? That Obstructs Nuclear Pore Complex Function ▿
Article Snippet: .. H68-GFP, H60-GFP, H68AA1-GFP, and H68AA2-GFP were expressed as N-terminal 6×His-tagged proteins from the pTriEx1 plasmid in Escherichia coli strain Turner(DE3)(pLysS) according to the manufacturer's instructions (Novagen). .. Cells were harvested by centrifugation and lysed in buffer containing 100 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1 mg/ml of lysozyme.

Article Title: The Effect of Transient Local Anti-inflammatory Treatment on the Survival of Pig Retinal Progenitor Cell Allotransplants
Article Snippet: .. Dual-label IHC was performed using a cocktail of anti-GFP (Abcam, ab13970 1:500) to detect GFP pRPCs and anti-Ki-67 (Vector, VP-K452 1:100; proliferation), anti-recoverin (Millipore, AB5585 1:500; cones), or mouse anti-rhodopsin (Millipore, MABN15 1:500; rods) antibodies. .. For infiltrating leukocytes/macrophages, we used anti-CD45 antibody (Abcam, 1:200).

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  • 96
    Millipore gö6983
    Acute activation or inhibition of PKC activity induces NMII remodeling. T cells (5C.C7) expressing MyoRLC-RFP were imaged in TIRF and treated with 5 ng/mL PMA or 500 nM <t>Gö6983</t> as indicated during time-lapse acquisition. ( A ) Representative time-lapse montages, with addition of reagents indicated by the red line. (Scale bars: 5 μm.) ( B – D ) Clustering of MyoRLC at the membrane was quantified by calculation of the SD of the fluorescence signals for each cell ( n ). The time of reagent addition is indicated by the gap in each curve. Error bars denote SEM.
    Gö6983, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore noc 7
    Depletion of kinesin-5 reduces the distance of axonal retraction. (A and B) DIC images of control axons (A) and kinesin-5–depleted axons (B) before and 30 min after treatment with 0.3 mM <t>noc-7.</t> (B) Kinesin-5–depleted axons continue to elongate even in the presence of noc-7. Arrows demarcate the distal tips of growth cones before and after noc-7 treatment. (C) Quantitative analysis of noc-7–induced axonal retraction revealed a statistically significant reduction in mean distance retracted in neurons depleted of kinesin-5 (mean ± SEM; control, n = 54; kinesin-5 siRNA, n = 55; *, P = 0.011). Bar, 20 μm.
    Noc 7, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Acute activation or inhibition of PKC activity induces NMII remodeling. T cells (5C.C7) expressing MyoRLC-RFP were imaged in TIRF and treated with 5 ng/mL PMA or 500 nM Gö6983 as indicated during time-lapse acquisition. ( A ) Representative time-lapse montages, with addition of reagents indicated by the red line. (Scale bars: 5 μm.) ( B – D ) Clustering of MyoRLC at the membrane was quantified by calculation of the SD of the fluorescence signals for each cell ( n ). The time of reagent addition is indicated by the gap in each curve. Error bars denote SEM.

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

    Article Title: Diacylglycerol promotes centrosome polarization in T cells via reciprocal localization of dynein and myosin II

    doi: 10.1073/pnas.1306180110

    Figure Lengend Snippet: Acute activation or inhibition of PKC activity induces NMII remodeling. T cells (5C.C7) expressing MyoRLC-RFP were imaged in TIRF and treated with 5 ng/mL PMA or 500 nM Gö6983 as indicated during time-lapse acquisition. ( A ) Representative time-lapse montages, with addition of reagents indicated by the red line. (Scale bars: 5 μm.) ( B – D ) Clustering of MyoRLC at the membrane was quantified by calculation of the SD of the fluorescence signals for each cell ( n ). The time of reagent addition is indicated by the gap in each curve. Error bars denote SEM.

    Article Snippet: When necessary, cells were incubated with PMA (5 ng/mL; Sigma), Gö6983 (500 nM; Calbiochem), blebbistatin (50 μM; Sigma), and Y-27632 (50 μM; Sigma) for 10 min before imaging.

    Techniques: Activation Assay, Inhibition, Activity Assay, Expressing, Fluorescence

    PI3K and PKD activities mediate BCR-dependent CXCR4/CXCR5 down-regulation but not CD62L release in CLL cells CLL cells were incubated for 24 hours in the presence (anti-IgM) or not (Unstimulated) of immobilized anti-IgM antibodies and in the presence or not (Untreated) of A. Idelalisib (50 μM), B. C. Gö6983 (1 μM), Gö6976 (1 μM), GF109203X (1 μM) or D. CID755673 (50 μM). CXCR4 and CD19 expressions were determined by flow cytometry. A. - C. Left panels show a representative sample for each treatment. Right panels depict the percentage of CXCR4 decrease upon stimulation that was calculated (cf. material and methods) and graphed from various CLL samples. D. shows BCR-mediated CXCR4 (left panel), CXCR5 (middle panel) and CD62L (right panel) decreases with (CID755673) or without (Untreated) PKD inhibitor. ** p

    Journal: Oncotarget

    Article Title: Protein kinase D-dependent CXCR4 down-regulation upon BCR triggering is linked to lymphadenopathy in chronic lymphocytic leukaemia

    doi: 10.18632/oncotarget.9031

    Figure Lengend Snippet: PI3K and PKD activities mediate BCR-dependent CXCR4/CXCR5 down-regulation but not CD62L release in CLL cells CLL cells were incubated for 24 hours in the presence (anti-IgM) or not (Unstimulated) of immobilized anti-IgM antibodies and in the presence or not (Untreated) of A. Idelalisib (50 μM), B. C. Gö6983 (1 μM), Gö6976 (1 μM), GF109203X (1 μM) or D. CID755673 (50 μM). CXCR4 and CD19 expressions were determined by flow cytometry. A. - C. Left panels show a representative sample for each treatment. Right panels depict the percentage of CXCR4 decrease upon stimulation that was calculated (cf. material and methods) and graphed from various CLL samples. D. shows BCR-mediated CXCR4 (left panel), CXCR5 (middle panel) and CD62L (right panel) decreases with (CID755673) or without (Untreated) PKD inhibitor. ** p

    Article Snippet: For BCR stimulation, CLL B cells (4×106 cells/well/12-well plate) were incubated with immobilized rabbit anti-IgM antibody (10 μg/mL; Jackson Immunoresearch, Montlucon, France) and incubated or not with Gö6976, Gö6983 (Calbiochem, Saint-Quentin-en-Yvellines, France), GF109203X, LY294002 (Sigma-Aldrich, Saint-Quentin-Fallavier, France), CAL101 (Selleckchem, Souffelweyersheim, France), CID755673 (Tocris Bioscience, Lille, France) or phorbol 12-myristate 13-acetate (PMA) (Cell Signaling Technology, Saint-Quentin-Fallavier, France).

    Techniques: Incubation, Flow Cytometry, Cytometry

    Effect of PKC inhibitors on THP-1 cells synthesis of proMMP-9/CSPG heteromer and proMMP-9. Shown is a typical experiment where cells in the absence (−) and presence (+) of PMA (10 −7 M) were incubated with various concentrations of the PKC inhibitors Gö6976 and Gö6983 ( A, B ) and Rottlerin ( C, D ). In the presence of Gö6976 and Gö6983 ( A, B ), cells were incubated for 72 h, while in the presence of Rottlerin ( C, D ) the cells were incubated for 12 h in serum free medium. To detect the effect of the PKC inhibitors on the synthesis of the proMMP-9/CSPG heteromer ( A, C ), the harvested media was applied to Q-Sepharose chromatography as described in Materials and Methods prior to gelatin zymography. To determine the effect of the inhibitors on the synthesised proMMP-9 ( B, D ), the harvested medium was diluted 20 times and then applied to gelatin zymography. Arrowhead shows the border between the separating and stacking gel and the position of purified proMMP-9 monomer (92 kDa) and proMMP-9 homodimer (225 kDa) used as a standard (Std) is shown.

    Journal: PLoS ONE

    Article Title: Biosynthesis of Promatrix Metalloproteinase-9/Chondroitin Sulphate Proteoglycan Heteromer Involves a Rottlerin-Sensitive Pathway

    doi: 10.1371/journal.pone.0020616

    Figure Lengend Snippet: Effect of PKC inhibitors on THP-1 cells synthesis of proMMP-9/CSPG heteromer and proMMP-9. Shown is a typical experiment where cells in the absence (−) and presence (+) of PMA (10 −7 M) were incubated with various concentrations of the PKC inhibitors Gö6976 and Gö6983 ( A, B ) and Rottlerin ( C, D ). In the presence of Gö6976 and Gö6983 ( A, B ), cells were incubated for 72 h, while in the presence of Rottlerin ( C, D ) the cells were incubated for 12 h in serum free medium. To detect the effect of the PKC inhibitors on the synthesis of the proMMP-9/CSPG heteromer ( A, C ), the harvested media was applied to Q-Sepharose chromatography as described in Materials and Methods prior to gelatin zymography. To determine the effect of the inhibitors on the synthesised proMMP-9 ( B, D ), the harvested medium was diluted 20 times and then applied to gelatin zymography. Arrowhead shows the border between the separating and stacking gel and the position of purified proMMP-9 monomer (92 kDa) and proMMP-9 homodimer (225 kDa) used as a standard (Std) is shown.

    Article Snippet: MCSF (macrophage colony stimulating factor), TNF-α, bFGF, IL-3, IL-6, Gö6983, Gö6976 and Rottlerin were from Calbiochem.

    Techniques: Incubation, Chromatography, Zymography, Purification

    Depletion of kinesin-5 reduces the distance of axonal retraction. (A and B) DIC images of control axons (A) and kinesin-5–depleted axons (B) before and 30 min after treatment with 0.3 mM noc-7. (B) Kinesin-5–depleted axons continue to elongate even in the presence of noc-7. Arrows demarcate the distal tips of growth cones before and after noc-7 treatment. (C) Quantitative analysis of noc-7–induced axonal retraction revealed a statistically significant reduction in mean distance retracted in neurons depleted of kinesin-5 (mean ± SEM; control, n = 54; kinesin-5 siRNA, n = 55; *, P = 0.011). Bar, 20 μm.

    Journal: The Journal of Cell Biology

    Article Title: Kinesin-5 regulates the growth of the axon by acting as a brake on its microtubule array

    doi: 10.1083/jcb.200702074

    Figure Lengend Snippet: Depletion of kinesin-5 reduces the distance of axonal retraction. (A and B) DIC images of control axons (A) and kinesin-5–depleted axons (B) before and 30 min after treatment with 0.3 mM noc-7. (B) Kinesin-5–depleted axons continue to elongate even in the presence of noc-7. Arrows demarcate the distal tips of growth cones before and after noc-7 treatment. (C) Quantitative analysis of noc-7–induced axonal retraction revealed a statistically significant reduction in mean distance retracted in neurons depleted of kinesin-5 (mean ± SEM; control, n = 54; kinesin-5 siRNA, n = 55; *, P = 0.011). Bar, 20 μm.

    Article Snippet: DIC images of axons were recorded before and 30 min after addition of noc-7.

    Techniques: