Structured Review

Qiagen hmec 1 cells
DLL4 and JAG1 induced endogenous JAG1 expression in tumour tissues A . Expression of JAG1 (red) and CD31 (green) as revealed by immunofluorescence double staining of tumour sections of U87. Magnification 200X. B . Endogenous hJAG1 expression in parental U87 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental U87 cells. hJAG1 expression in GFP-negative U87 cells was detected by FACS staining with anti-human JAG1 mAb (64D). ANOVA with Bonferroni's post-test. N = 3, Error bars represent SD. C . Endogenous hJAG1 expression in parental <t>HMEC-1</t> cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental HMEC-1 cells. hJAG1 expression in GFP-negative HMEC-1 cells was detected by FACS staining with anti-human JAG1 mAb (64D). ANOVA with Bonferroni's post-test. NS, no statistical difference. N = 3, Error bars represent SD. * P
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1) Product Images from "Role of Delta-like 4 in Jagged1-induced tumour angiogenesis and tumour growth"

Article Title: Role of Delta-like 4 in Jagged1-induced tumour angiogenesis and tumour growth

Journal: Oncotarget

doi: 10.18632/oncotarget.16969

DLL4 and JAG1 induced endogenous JAG1 expression in tumour tissues A . Expression of JAG1 (red) and CD31 (green) as revealed by immunofluorescence double staining of tumour sections of U87. Magnification 200X. B . Endogenous hJAG1 expression in parental U87 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental U87 cells. hJAG1 expression in GFP-negative U87 cells was detected by FACS staining with anti-human JAG1 mAb (64D). ANOVA with Bonferroni's post-test. N = 3, Error bars represent SD. C . Endogenous hJAG1 expression in parental HMEC-1 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental HMEC-1 cells. hJAG1 expression in GFP-negative HMEC-1 cells was detected by FACS staining with anti-human JAG1 mAb (64D). ANOVA with Bonferroni's post-test. NS, no statistical difference. N = 3, Error bars represent SD. * P
Figure Legend Snippet: DLL4 and JAG1 induced endogenous JAG1 expression in tumour tissues A . Expression of JAG1 (red) and CD31 (green) as revealed by immunofluorescence double staining of tumour sections of U87. Magnification 200X. B . Endogenous hJAG1 expression in parental U87 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental U87 cells. hJAG1 expression in GFP-negative U87 cells was detected by FACS staining with anti-human JAG1 mAb (64D). ANOVA with Bonferroni's post-test. N = 3, Error bars represent SD. C . Endogenous hJAG1 expression in parental HMEC-1 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental HMEC-1 cells. hJAG1 expression in GFP-negative HMEC-1 cells was detected by FACS staining with anti-human JAG1 mAb (64D). ANOVA with Bonferroni's post-test. NS, no statistical difference. N = 3, Error bars represent SD. * P

Techniques Used: Expressing, Immunofluorescence, Double Staining, Co-Culture Assay, FACS, Staining

DLL4 and JAG1 induced endogenous DLL4 expression in tumour vessels A . Expression of DLL4 (red) and CD31 (green) as revealed by immunofluorescence double staining of tumour sections of U87. Magnification 200X. B . Endogenous hDLL4 expression in parental U87 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental U87 cells. hDLL4 expression in GFP-negative U87 cells was detected by FACS staining with anti-DLL4 antibody (recognised both human and mouse DLL4). ANOVA with Bonferroni's post-test. NS, no statistical difference. N = 3. Error bars represent SD. C . Endogenous hDLL4 expression in parental HMEC-1 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental HMEC-1 cells. hDLL4 expression in GFP-negative HMEC-1 cells was detected by FACS staining with anti-DLL4 antibody. ANOVA with Bonferroni's post-test. NS, no statistical difference. N = 3, Error bars represent SD. * P
Figure Legend Snippet: DLL4 and JAG1 induced endogenous DLL4 expression in tumour vessels A . Expression of DLL4 (red) and CD31 (green) as revealed by immunofluorescence double staining of tumour sections of U87. Magnification 200X. B . Endogenous hDLL4 expression in parental U87 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental U87 cells. hDLL4 expression in GFP-negative U87 cells was detected by FACS staining with anti-DLL4 antibody (recognised both human and mouse DLL4). ANOVA with Bonferroni's post-test. NS, no statistical difference. N = 3. Error bars represent SD. C . Endogenous hDLL4 expression in parental HMEC-1 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental HMEC-1 cells. hDLL4 expression in GFP-negative HMEC-1 cells was detected by FACS staining with anti-DLL4 antibody. ANOVA with Bonferroni's post-test. NS, no statistical difference. N = 3, Error bars represent SD. * P

Techniques Used: Expressing, Immunofluorescence, Double Staining, Co-Culture Assay, FACS, Staining

DLL4 and JAG1 activated Notch signalling and affected sprouting angiogenesis in vitro . A . Expression profile of Notch target genes in HMEC-1 stimulated with rhDLL4 or rrJAG1 over a time course. QPCR was used to determine mRNA levels of DLL4, JAG1, HEY1 and HEY2. Fold changes were obtained by normalizing against the EV control. B . Expression profile of Notch target genes in HMEC-1 over-expressing mDLL4 or mJAG1 by retrovirus transductions. QPCR was used to determine mRNA levels of DLL4, JAG1, HEY1 and HEY2 (ANOVA with Bonferroni's post-test). C . Expression of endogenous hDLL4 and hJAG1 proteins in parental HMEC-1 cells (GFP-negative) sorted from co-culture of EV-, mDLL4- or mJAG1-overexpressing HMEC-1 with an equal amount of parental HMEC-1 cells by FACS analysis (ANOVA with Bonferroni's post-test). D . Effect of mDLL4 and mJAG1 expressed in HMEC-1 cells on sprouting in HMEC-1 spheroids and treated with DBZ. Average lengths of three longest sprouts were calculated for statistical test (ANOVA with Bonferroni's post-test). Graphs are means of 3 independent experiments. E . Effect of knockdown of DLL4 in HMEC-1 cells by specific siDLL4 on sprouting in HMEC-1 spheroids. Average lengths of three longest sprouts were calculated for statistical test (ANOVA with Bonferroni's post-test). Graphs are means of 3 independent experiments. F . Effect of knockdown of JAG1 in HMEC-1 cells by specific siJAG1 on sprouting in HMEC-1 spheroids. Average lengths of three longest sprouts were calculated for statistical test (ANOVA with Bonferroni's post-test). Graphs are means of 3 independent experiments. * P
Figure Legend Snippet: DLL4 and JAG1 activated Notch signalling and affected sprouting angiogenesis in vitro . A . Expression profile of Notch target genes in HMEC-1 stimulated with rhDLL4 or rrJAG1 over a time course. QPCR was used to determine mRNA levels of DLL4, JAG1, HEY1 and HEY2. Fold changes were obtained by normalizing against the EV control. B . Expression profile of Notch target genes in HMEC-1 over-expressing mDLL4 or mJAG1 by retrovirus transductions. QPCR was used to determine mRNA levels of DLL4, JAG1, HEY1 and HEY2 (ANOVA with Bonferroni's post-test). C . Expression of endogenous hDLL4 and hJAG1 proteins in parental HMEC-1 cells (GFP-negative) sorted from co-culture of EV-, mDLL4- or mJAG1-overexpressing HMEC-1 with an equal amount of parental HMEC-1 cells by FACS analysis (ANOVA with Bonferroni's post-test). D . Effect of mDLL4 and mJAG1 expressed in HMEC-1 cells on sprouting in HMEC-1 spheroids and treated with DBZ. Average lengths of three longest sprouts were calculated for statistical test (ANOVA with Bonferroni's post-test). Graphs are means of 3 independent experiments. E . Effect of knockdown of DLL4 in HMEC-1 cells by specific siDLL4 on sprouting in HMEC-1 spheroids. Average lengths of three longest sprouts were calculated for statistical test (ANOVA with Bonferroni's post-test). Graphs are means of 3 independent experiments. F . Effect of knockdown of JAG1 in HMEC-1 cells by specific siJAG1 on sprouting in HMEC-1 spheroids. Average lengths of three longest sprouts were calculated for statistical test (ANOVA with Bonferroni's post-test). Graphs are means of 3 independent experiments. * P

Techniques Used: In Vitro, Expressing, Real-time Polymerase Chain Reaction, Co-Culture Assay, FACS

2) Product Images from "Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin"

Article Title: Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E18-04-0228

Surface vimentin is localized along the periphery of HMEC-1. (A–D) Cells were stained for vimentin using the rabbit anti-vimentin H-84 antibody. For negative controls, cells were stained with secondary anti-rabbit IgG antibody alone. Representative phase image of cells (left column), image of the nuclei (middle column), and H-84 anti-vimentin antibody fluorescence (right column) are shown for (A) permeabilized HMEC-1 strained for intracellular vimentin; (B) permeabilized HMEC-1 incubated with anti-rabbit IgG alone as negative controls; (C) nonpermeabilized HMEC-1 stained for surface vimentin; (D) nonpermeabilized HMEC-1 incubated with anti-rabbit IgG alone as negative controls. Scale bar shown in white is 20 μm. White arrows point at the localization of surface vimentin at cell–cell junctions.
Figure Legend Snippet: Surface vimentin is localized along the periphery of HMEC-1. (A–D) Cells were stained for vimentin using the rabbit anti-vimentin H-84 antibody. For negative controls, cells were stained with secondary anti-rabbit IgG antibody alone. Representative phase image of cells (left column), image of the nuclei (middle column), and H-84 anti-vimentin antibody fluorescence (right column) are shown for (A) permeabilized HMEC-1 strained for intracellular vimentin; (B) permeabilized HMEC-1 incubated with anti-rabbit IgG alone as negative controls; (C) nonpermeabilized HMEC-1 stained for surface vimentin; (D) nonpermeabilized HMEC-1 incubated with anti-rabbit IgG alone as negative controls. Scale bar shown in white is 20 μm. White arrows point at the localization of surface vimentin at cell–cell junctions.

Techniques Used: Staining, Fluorescence, Incubation

Infection of HMEC-1 by Lm is in part mediated by InlB in a manner independent of matrix stiffness. (A) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: wild type (circle); Δ inlA (square); Δ inlB (cross); Δ inlF (diamond; actAp::mTagRFP). The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. (B) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: Δ actA (black circles); Δ actA/ Δ inlB (gray squares; actAp::mTagRFP). The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. (C) Boxplots of percentage of HMEC-1 infected with Lm as a function of substrate stiffness ( N = 5–6 replicates). HMEC-1 were infected with the indicated Lm strains: Δ actA (gray); Δ actA/ Δ inlB (black; actAp::mTagRFP) at an MOI of 20. Infection was analyzed by flow cytometry 7–8 h after infection. Representative data come from one of three independent experiments. One or two asterisks denote statistically significant differences between the medians of two distributions (
Figure Legend Snippet: Infection of HMEC-1 by Lm is in part mediated by InlB in a manner independent of matrix stiffness. (A) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: wild type (circle); Δ inlA (square); Δ inlB (cross); Δ inlF (diamond; actAp::mTagRFP). The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. (B) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: Δ actA (black circles); Δ actA/ Δ inlB (gray squares; actAp::mTagRFP). The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. (C) Boxplots of percentage of HMEC-1 infected with Lm as a function of substrate stiffness ( N = 5–6 replicates). HMEC-1 were infected with the indicated Lm strains: Δ actA (gray); Δ actA/ Δ inlB (black; actAp::mTagRFP) at an MOI of 20. Infection was analyzed by flow cytometry 7–8 h after infection. Representative data come from one of three independent experiments. One or two asterisks denote statistically significant differences between the medians of two distributions (

Techniques Used: Infection, Flow Cytometry, Cytometry

Uptake of Lm by HMEC-1 depends on the stiffness of the matrix on which cells reside. HMEC-1 residing on PA hydrogels of varying stiffness coated with collagen I were infected with Δ actA Lm (actAp::mTagRFP). Infection was analyzed by flow cytometry 7–8 h postinfection. Bacteria were added at a multiplicity of infection (MOI) between 30 and 50 bacteria per host cell. (A–D) Histograms of the logarithm of bacterial fluorescence intensity per cell for HMEC-1 plated on 0.6-kPa (A), 3-kPa (B), 20-kPa (C), and 70-kPa (D) PA hydrogels. Histograms for N = 5 replicates are shown in different colors. The histogram of control uninfected cells is shown in purple. Based on the autofluorescence of the control group, a gate is defined (see black and red lines) showing what is considered uninfected (left, black line) and infected (right, red line). (E) Boxplots of percentage of HMEC-1 infected with Δ actA Lm vs. hydrogel stiffness for the data shown in panels A–D. Circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test; for details about boxplots see Materials and Methods ). One or two asterisks denote statistically significant differences between the medians of two distributions (
Figure Legend Snippet: Uptake of Lm by HMEC-1 depends on the stiffness of the matrix on which cells reside. HMEC-1 residing on PA hydrogels of varying stiffness coated with collagen I were infected with Δ actA Lm (actAp::mTagRFP). Infection was analyzed by flow cytometry 7–8 h postinfection. Bacteria were added at a multiplicity of infection (MOI) between 30 and 50 bacteria per host cell. (A–D) Histograms of the logarithm of bacterial fluorescence intensity per cell for HMEC-1 plated on 0.6-kPa (A), 3-kPa (B), 20-kPa (C), and 70-kPa (D) PA hydrogels. Histograms for N = 5 replicates are shown in different colors. The histogram of control uninfected cells is shown in purple. Based on the autofluorescence of the control group, a gate is defined (see black and red lines) showing what is considered uninfected (left, black line) and infected (right, red line). (E) Boxplots of percentage of HMEC-1 infected with Δ actA Lm vs. hydrogel stiffness for the data shown in panels A–D. Circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test; for details about boxplots see Materials and Methods ). One or two asterisks denote statistically significant differences between the medians of two distributions (

Techniques Used: Infection, Flow Cytometry, Cytometry, Fluorescence, MANN-WHITNEY

Lm adhesion, but not invasion efficiency, is increased when HMEC-1 reside on stiff hydrogels. HMEC-1 residing on soft (3-kPa) or stiff (70-kPa) PA hydrogels and treated with vehicle control or 2 μM PF537228 FAK inhibitor were infected with Lm (constitutively expressing GFP) at an MOI between 1.5 and 15. At 30 min postinfection, samples were fixed and immunostained, and infection was analyzed by microscopy followed by image processing. Boxplots show (A) total bacteria per cell; (B) internalized bacteria per cell; (C) invasion efficiency (ratio of internalized bacteria to total bacteria); (D) cells in the field of view. Representative data come from one of three independent experiments. N = 800–1000 cells were analyzed for each condition. Two asterisks denote statistically significant differences between the medians of two distributions (
Figure Legend Snippet: Lm adhesion, but not invasion efficiency, is increased when HMEC-1 reside on stiff hydrogels. HMEC-1 residing on soft (3-kPa) or stiff (70-kPa) PA hydrogels and treated with vehicle control or 2 μM PF537228 FAK inhibitor were infected with Lm (constitutively expressing GFP) at an MOI between 1.5 and 15. At 30 min postinfection, samples were fixed and immunostained, and infection was analyzed by microscopy followed by image processing. Boxplots show (A) total bacteria per cell; (B) internalized bacteria per cell; (C) invasion efficiency (ratio of internalized bacteria to total bacteria); (D) cells in the field of view. Representative data come from one of three independent experiments. N = 800–1000 cells were analyzed for each condition. Two asterisks denote statistically significant differences between the medians of two distributions (

Techniques Used: Infection, Expressing, Microscopy

Lower FAK activity leads to reduced amount of cell surface vimentin. (A, B) 2D-PAGE gels of plasma membrane proteins of HMEC-1 grown on TC polystyrene substrates treated for 1 h with vehicle control (A) or 2 µM PF537228 FAK inhibitor (B). pH increases from left to right. Gels were silver-stained and one isoelectric point marker (tropomyosin), added to each sample as an internal standard, is marked with a black arrow. The one spot that differed consistently between three independent experiments is indicated with a black circle and corresponds to vimentin (55 kDa).
Figure Legend Snippet: Lower FAK activity leads to reduced amount of cell surface vimentin. (A, B) 2D-PAGE gels of plasma membrane proteins of HMEC-1 grown on TC polystyrene substrates treated for 1 h with vehicle control (A) or 2 µM PF537228 FAK inhibitor (B). pH increases from left to right. Gels were silver-stained and one isoelectric point marker (tropomyosin), added to each sample as an internal standard, is marked with a black arrow. The one spot that differed consistently between three independent experiments is indicated with a black circle and corresponds to vimentin (55 kDa).

Techniques Used: Activity Assay, Polyacrylamide Gel Electrophoresis, Staining, Marker

Blocking HMEC-1 with anti-vimentin antibody reduces Li adhesion onto HMEC-1 but not uptake of beads. (A) Boxplots showing the number of bacteria per cell, for HMEC-1 residing on glass substrates and treated with vehicle control, 2 μM PF537228 FAK inhibitor, or 80 μg/ml H-84 anti-vimentin antibody prior to infection. Cells were infected with Lm or Li at an MOI of 4. At 30 min postinfection, samples were fixed and immunostained and adhesion of bacteria was analyzed by microscopy followed by image processing. For each condition, 2300–2600 cells were analyzed in total and data refer to one of two independent experiments. Two asterisks denote statistically significant differences between the median values of control cells vs. all other groups (
Figure Legend Snippet: Blocking HMEC-1 with anti-vimentin antibody reduces Li adhesion onto HMEC-1 but not uptake of beads. (A) Boxplots showing the number of bacteria per cell, for HMEC-1 residing on glass substrates and treated with vehicle control, 2 μM PF537228 FAK inhibitor, or 80 μg/ml H-84 anti-vimentin antibody prior to infection. Cells were infected with Lm or Li at an MOI of 4. At 30 min postinfection, samples were fixed and immunostained and adhesion of bacteria was analyzed by microscopy followed by image processing. For each condition, 2300–2600 cells were analyzed in total and data refer to one of two independent experiments. Two asterisks denote statistically significant differences between the median values of control cells vs. all other groups (

Techniques Used: Blocking Assay, Infection, Microscopy

FAK activity of HMEC-1 residing on soft PA hydrogels is decreased, as is Lm uptake. (A) Western blots from whole HMEC-1 lysates showing expression of phosphorylated FAK (Tyr397) and total FAK for cells residing on soft gels (3 kPa), stiff gels (70 kPa), and TC polystyrene substrates with or without 2 μM PF537228 FAK inhibitor. In each Western blot, equal quantities of protein were loaded and equal loading was confirmed in relation to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. In each case, the Western blots shown are representative of three independent experiments. (B, C) Normalized ratios of FAK/GAPDH (B) and pFAK (Tyr397)/GAPDH (C) for HMEC-1 residing on varying-stiffness substrates and treated or not with 2 μM PF537228 FAK inhibitor. Different color circles correspond to data from three independent experiments. Black bars represent the means of the three independent experiments. For each experiment, values have been normalized relative to the ratio for cells residing on polystyrene substrates. (D) Inhibition of bacterial uptake by FAK inhibitors. FAK-14, PF573228, or vehicle control was added 1 h before addition of bacteria to HMEC-1 residing on polystyrene substrates. Percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of inhibitor concentration (mean ± SD, N = 4 replicates). x = 0 corresponds to cells treated with vehicle control. Inset shows the same data with concentration on a log scale. Infection was analyzed by flow cytometry, 7–8 h after infection. MOI is 80. Representative data come from one of three independent experiments. (E) Boxplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or FAK siRNA (siFAK) (means ± SD, three independent experiments and N = 6 replicates per experiment). MOI is 60 (gray) or 20 (green). Circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test; for details about boxplots see Materials and Methods ). One or two asterisks denote statistically significant differences between the medians of two distributions (
Figure Legend Snippet: FAK activity of HMEC-1 residing on soft PA hydrogels is decreased, as is Lm uptake. (A) Western blots from whole HMEC-1 lysates showing expression of phosphorylated FAK (Tyr397) and total FAK for cells residing on soft gels (3 kPa), stiff gels (70 kPa), and TC polystyrene substrates with or without 2 μM PF537228 FAK inhibitor. In each Western blot, equal quantities of protein were loaded and equal loading was confirmed in relation to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. In each case, the Western blots shown are representative of three independent experiments. (B, C) Normalized ratios of FAK/GAPDH (B) and pFAK (Tyr397)/GAPDH (C) for HMEC-1 residing on varying-stiffness substrates and treated or not with 2 μM PF537228 FAK inhibitor. Different color circles correspond to data from three independent experiments. Black bars represent the means of the three independent experiments. For each experiment, values have been normalized relative to the ratio for cells residing on polystyrene substrates. (D) Inhibition of bacterial uptake by FAK inhibitors. FAK-14, PF573228, or vehicle control was added 1 h before addition of bacteria to HMEC-1 residing on polystyrene substrates. Percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of inhibitor concentration (mean ± SD, N = 4 replicates). x = 0 corresponds to cells treated with vehicle control. Inset shows the same data with concentration on a log scale. Infection was analyzed by flow cytometry, 7–8 h after infection. MOI is 80. Representative data come from one of three independent experiments. (E) Boxplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or FAK siRNA (siFAK) (means ± SD, three independent experiments and N = 6 replicates per experiment). MOI is 60 (gray) or 20 (green). Circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test; for details about boxplots see Materials and Methods ). One or two asterisks denote statistically significant differences between the medians of two distributions (

Techniques Used: Activity Assay, Western Blot, Expressing, Inhibition, Infection, Concentration Assay, Flow Cytometry, Cytometry, MANN-WHITNEY

Surface vimentin of HMEC-1 is implicated in Lm uptake. (A) Decrease in bacterial uptake after blocking HMEC-1 with anti-vimentin antibody H-84. Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of antibody concentration (means ± SD and N = 6 replicates per experiment). Representative data come from one of three independent experiments. Infection was analyzed by flow cytometry, 7–8 h after infection. (B) Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or with vimentin siRNA (siVIM) (means ± SD, and N = 6 replicates per experiment). Representative data come from one of three independent experiments. MOI is 50 (black barplots) and 17 (gray barplots). (C) Decreased uptake of Lm when HMEC-1 are treated with withaferin that captures soluble vimentin 30 min prior to infection. Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of withaferin concentration (means ± SD and N = 6 replicates per experiment). Representative data come from one of three independent experiments. Infection was analyzed by flow cytometry 7–8 h after infection. (D) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: Δ actA (black), Δ actA/ Δ inlB (gray; actAp::mTagRFP), and HMEC-1 were treated with vehicle control (circle) or withaferin (diamond) for 30 min prior to infection. The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. MOI ranged from 50 to120. Two asterisks denote statistically significant differences between the medians of infection fraction of control vs. all other groups ( p
Figure Legend Snippet: Surface vimentin of HMEC-1 is implicated in Lm uptake. (A) Decrease in bacterial uptake after blocking HMEC-1 with anti-vimentin antibody H-84. Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of antibody concentration (means ± SD and N = 6 replicates per experiment). Representative data come from one of three independent experiments. Infection was analyzed by flow cytometry, 7–8 h after infection. (B) Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or with vimentin siRNA (siVIM) (means ± SD, and N = 6 replicates per experiment). Representative data come from one of three independent experiments. MOI is 50 (black barplots) and 17 (gray barplots). (C) Decreased uptake of Lm when HMEC-1 are treated with withaferin that captures soluble vimentin 30 min prior to infection. Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of withaferin concentration (means ± SD and N = 6 replicates per experiment). Representative data come from one of three independent experiments. Infection was analyzed by flow cytometry 7–8 h after infection. (D) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: Δ actA (black), Δ actA/ Δ inlB (gray; actAp::mTagRFP), and HMEC-1 were treated with vehicle control (circle) or withaferin (diamond) for 30 min prior to infection. The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. MOI ranged from 50 to120. Two asterisks denote statistically significant differences between the medians of infection fraction of control vs. all other groups ( p

Techniques Used: Blocking Assay, Infection, Concentration Assay, Flow Cytometry, Cytometry

3) Product Images from "Sustained Expression of Homeobox D10 Inhibits Angiogenesis"

Article Title: Sustained Expression of Homeobox D10 Inhibits Angiogenesis

Journal: The American Journal of Pathology

doi:

HoxD10 impairs EC migration. a: Relative levels of HoxD10 mRNA and protein in control and HoxD10-transfected HMEC-1. Top : RT-PCR and the relative amounts of the 499-bp product corresponding to HoxD10 mRNA. The relative RNA loading is shown by the 390-bp band corresponding to 18s ribosomal RNA determined using competitive primers. Bottom : Western blotting of control and HoxD10-transfected HMEC-1 lysates probed with a polyclonal antibody against HoxD10. The arrow indicates the position of the HoxD10 protein. b: Relative migration of control or HoxD10-transfected HMEC-1 on membranes coated with 10 μg of collagen. Migration was assessed after 3 hours in the presence of 50 ng/ml of bFGF (▨) or 50 ng/ml VEGF (▪) or with no treatment (□). Each panel presents the mean of eight individual determinations and error bars show the SD. **, P
Figure Legend Snippet: HoxD10 impairs EC migration. a: Relative levels of HoxD10 mRNA and protein in control and HoxD10-transfected HMEC-1. Top : RT-PCR and the relative amounts of the 499-bp product corresponding to HoxD10 mRNA. The relative RNA loading is shown by the 390-bp band corresponding to 18s ribosomal RNA determined using competitive primers. Bottom : Western blotting of control and HoxD10-transfected HMEC-1 lysates probed with a polyclonal antibody against HoxD10. The arrow indicates the position of the HoxD10 protein. b: Relative migration of control or HoxD10-transfected HMEC-1 on membranes coated with 10 μg of collagen. Migration was assessed after 3 hours in the presence of 50 ng/ml of bFGF (▨) or 50 ng/ml VEGF (▪) or with no treatment (□). Each panel presents the mean of eight individual determinations and error bars show the SD. **, P

Techniques Used: Migration, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot

Up-regulation of endogenous HoxD10 and related changes in gene expression. a: Top : relative levels of HoxD10 and 18s ribosomal RNA in HMEC-1 cultured in the presence (+) or absence (−) of reconstituted BM as determined by RT-PCR. Western blot analysis of HoxD10 protein levels ( middle ) or cyclin D1 levels ( bottom ) in 40 μg of total cell lysates from HMEC-1 cultured for 24 hours in the absence (−) or presence of (+) of reconstituted BM. b: Top : Northern blot analysis of α3 integrin ( top band ) and uPAR ( bottom band ) steady-state mRNA levels in 7.5 μg of total RNA from cells cultured in the absence (−) or presence (+) of reconstituted BM. Bottom : Relative RNA loading for each sample as visualized by staining with ethidium bromide. c: Histogram shows relative levels of α3 integrin and uPAR mRNA levels in cells cultured in the absence (▪) or presence (▨) of reconstituted BM as determined by densitometric analysis and correction for total RNA levels in each sample.
Figure Legend Snippet: Up-regulation of endogenous HoxD10 and related changes in gene expression. a: Top : relative levels of HoxD10 and 18s ribosomal RNA in HMEC-1 cultured in the presence (+) or absence (−) of reconstituted BM as determined by RT-PCR. Western blot analysis of HoxD10 protein levels ( middle ) or cyclin D1 levels ( bottom ) in 40 μg of total cell lysates from HMEC-1 cultured for 24 hours in the absence (−) or presence of (+) of reconstituted BM. b: Top : Northern blot analysis of α3 integrin ( top band ) and uPAR ( bottom band ) steady-state mRNA levels in 7.5 μg of total RNA from cells cultured in the absence (−) or presence (+) of reconstituted BM. Bottom : Relative RNA loading for each sample as visualized by staining with ethidium bromide. c: Histogram shows relative levels of α3 integrin and uPAR mRNA levels in cells cultured in the absence (▪) or presence (▨) of reconstituted BM as determined by densitometric analysis and correction for total RNA levels in each sample.

Techniques Used: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot, Northern Blot, Staining

Changes in gene expression and migration in three-dimensional fibrin gels. a: Far left : migration of control-transfected HMEC-1 in three-dimensional fibrin gels without cytokine treatment (control). The next panel shows control-transfected HMEC-1 treated with 50 ng of bFGF (bFGF) or after the addition of 50 ng of bFGF and 25 μg of a function-blocking antibody against uPAR (bFGF+anti-uPAR). Far right: Relative migration of HoxD10-transfected HMEC-1 embedded within fibrin gels 48 hours after exposure to bFGF only (bFGF+HoxD10). b: Top : Northern blot analysis of uPAR mRNA expression in control- and HoxD10-expressing HMEC-1. Middle : Relative loading as visualized by staining of ribosomal RNA (rRNA) with ethidium bromide. Bottom : Western blot analysis for uPAR protein expression in whole cell lysates from control- or HoxD10-transfected HMEC-1. c: Relative levels of HoxD3 ( top ) or HoxB3 ( middle ) in control- or HoxD10-transfected HMEC-1 as determined by RT-PCR. Total mRNA for each sample was determined by RT-PCR using competitive primers against 18s ribosomal RNA in each sample ( bottom ). d: Northern blot analysis of β3 integrin ( top ) and ephrin A1 ( middle ) steady-state mRNA levels in control- and HoxD10-transfected HMEC-1. Total RNA in each sample is visualized by staining of ribosomal RNA with ethidium bromide ( bottom ). Original magnification, ×20 ( a ).
Figure Legend Snippet: Changes in gene expression and migration in three-dimensional fibrin gels. a: Far left : migration of control-transfected HMEC-1 in three-dimensional fibrin gels without cytokine treatment (control). The next panel shows control-transfected HMEC-1 treated with 50 ng of bFGF (bFGF) or after the addition of 50 ng of bFGF and 25 μg of a function-blocking antibody against uPAR (bFGF+anti-uPAR). Far right: Relative migration of HoxD10-transfected HMEC-1 embedded within fibrin gels 48 hours after exposure to bFGF only (bFGF+HoxD10). b: Top : Northern blot analysis of uPAR mRNA expression in control- and HoxD10-expressing HMEC-1. Middle : Relative loading as visualized by staining of ribosomal RNA (rRNA) with ethidium bromide. Bottom : Western blot analysis for uPAR protein expression in whole cell lysates from control- or HoxD10-transfected HMEC-1. c: Relative levels of HoxD3 ( top ) or HoxB3 ( middle ) in control- or HoxD10-transfected HMEC-1 as determined by RT-PCR. Total mRNA for each sample was determined by RT-PCR using competitive primers against 18s ribosomal RNA in each sample ( bottom ). d: Northern blot analysis of β3 integrin ( top ) and ephrin A1 ( middle ) steady-state mRNA levels in control- and HoxD10-transfected HMEC-1. Total RNA in each sample is visualized by staining of ribosomal RNA with ethidium bromide ( bottom ). Original magnification, ×20 ( a ).

Techniques Used: Expressing, Migration, Transfection, Blocking Assay, Northern Blot, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction

HoxD10 blocks angiogenesis of human ECs implanted into nu/nu mice. a: CD34 staining of a 7-μm section of tissue adjacent to implanted PVA sponges seeded with HMEC-1 recovered from a nude mouse after 10 days. Arrows indicate the presence of small CD34-positive vessels. b: Higher magnification of a , showing microvessels consisting of CD34-positive human ECs surrounding red blood cells. c: CD34 staining of 7-μm section of tissue adjacent to PVA sponge (S) seeded with HoxD10-transfected HMEC-1 and recovered from nude mice 10 days after implantation. d: Higher magnification of c showing little or no positive staining for human CD34-derived microvessels in tissues adjacent to the sponge. Original magnifications: ×10 ( a , c ); ×40 ( b , d ).
Figure Legend Snippet: HoxD10 blocks angiogenesis of human ECs implanted into nu/nu mice. a: CD34 staining of a 7-μm section of tissue adjacent to implanted PVA sponges seeded with HMEC-1 recovered from a nude mouse after 10 days. Arrows indicate the presence of small CD34-positive vessels. b: Higher magnification of a , showing microvessels consisting of CD34-positive human ECs surrounding red blood cells. c: CD34 staining of 7-μm section of tissue adjacent to PVA sponge (S) seeded with HoxD10-transfected HMEC-1 and recovered from nude mice 10 days after implantation. d: Higher magnification of c showing little or no positive staining for human CD34-derived microvessels in tissues adjacent to the sponge. Original magnifications: ×10 ( a , c ); ×40 ( b , d ).

Techniques Used: Mouse Assay, Staining, Transfection, Derivative Assay

4) Product Images from "The ?3-Integrin Binding Protein ?3-Endonexin Is a Novel Negative Regulator of Hypoxia-Inducible Factor-1"

Article Title: The ?3-Integrin Binding Protein ?3-Endonexin Is a Novel Negative Regulator of Hypoxia-Inducible Factor-1

Journal: Antioxidants & Redox Signaling

doi: 10.1089/ars.2013.5286

β3-endonexin regulates hypoxic HIF-1 α transcription via NF κ B. (A) HMEC-1 cells were transfected with HIF-1 α promoter luciferase constructs containing either the wild-type NF κ B-binding site (HIF1 α -538) or a mutated NF κ B-binding site (HIF1 α -538m) and with siRNA against β3-endonexin (siEN) or scrambled RNA (siCtr) or with expression vectors coding for either β3-endonexin short (EN-S) or long (EN-L) isoforms or with control vector (Ctr). Cells were exposed to hypoxia (1% oxygen, Hx) for 8 h or remained under normoxia (Ctr), and luciferase assays were performed. Normoxic controls were set equal to 100% ( n =3, * p
Figure Legend Snippet: β3-endonexin regulates hypoxic HIF-1 α transcription via NF κ B. (A) HMEC-1 cells were transfected with HIF-1 α promoter luciferase constructs containing either the wild-type NF κ B-binding site (HIF1 α -538) or a mutated NF κ B-binding site (HIF1 α -538m) and with siRNA against β3-endonexin (siEN) or scrambled RNA (siCtr) or with expression vectors coding for either β3-endonexin short (EN-S) or long (EN-L) isoforms or with control vector (Ctr). Cells were exposed to hypoxia (1% oxygen, Hx) for 8 h or remained under normoxia (Ctr), and luciferase assays were performed. Normoxic controls were set equal to 100% ( n =3, * p

Techniques Used: Transfection, Luciferase, Construct, Binding Assay, Expressing, Plasmid Preparation

β3-endonexin modulates NF κ B activity. (A) HMEC-1 cells were co-transfected with a luciferase construct driven by five NF κ B binding-sites (NF κ B-Luc) and expression vectors coding for either β3-endonexin short (EN-S) or long (EN-L) isoforms or with control vector (Ctr) or with siRNA against β3-endonexin (siEN) or scrambled RNA (siCtr). Cells were exposed to hypoxia (1% oxygen, Hx) for 8 h or remained at normoxia (Ctr), and luciferase assays were performed. Normoxic controls were set equal to 100% ( n =3, * p
Figure Legend Snippet: β3-endonexin modulates NF κ B activity. (A) HMEC-1 cells were co-transfected with a luciferase construct driven by five NF κ B binding-sites (NF κ B-Luc) and expression vectors coding for either β3-endonexin short (EN-S) or long (EN-L) isoforms or with control vector (Ctr) or with siRNA against β3-endonexin (siEN) or scrambled RNA (siCtr). Cells were exposed to hypoxia (1% oxygen, Hx) for 8 h or remained at normoxia (Ctr), and luciferase assays were performed. Normoxic controls were set equal to 100% ( n =3, * p

Techniques Used: Activity Assay, Transfection, Luciferase, Construct, Binding Assay, Expressing, Plasmid Preparation

β3-endonexin accumulates in the nucleus under hypoxia. (A/B) HMEC-1 cells were seeded on uncoated coverslips (A) or on coverslips coated with collagen or fibronectin (B) and exposed to hypoxia (Hx) or normoxia (0 h) for increasing time periods. Cells were fixed, and immunostaining was performed with an antibody against β3-endonexin and a secondary antibody conjugated with Alexa fluor 488. Nuclei were visualized by 4′, 6-diamidino-2-phenylindole (DAPI) staining. Scale bars represent 10 μm. (C) HMEC-1 cells were exposed to hypoxia (Hx) for 8 h or remained under normoxia (0 h). Western blot analyses were performed on nuclear and cytosolic fractions with antibodies against HIF-1 α
Figure Legend Snippet: β3-endonexin accumulates in the nucleus under hypoxia. (A/B) HMEC-1 cells were seeded on uncoated coverslips (A) or on coverslips coated with collagen or fibronectin (B) and exposed to hypoxia (Hx) or normoxia (0 h) for increasing time periods. Cells were fixed, and immunostaining was performed with an antibody against β3-endonexin and a secondary antibody conjugated with Alexa fluor 488. Nuclei were visualized by 4′, 6-diamidino-2-phenylindole (DAPI) staining. Scale bars represent 10 μm. (C) HMEC-1 cells were exposed to hypoxia (Hx) for 8 h or remained under normoxia (0 h). Western blot analyses were performed on nuclear and cytosolic fractions with antibodies against HIF-1 α

Techniques Used: Immunostaining, Staining, Western Blot

β3-endonexin decreases hypoxic tube formation. (A) HMEC-1 cells were transfected with expression vectors coding for either β3-endonexin short (EN-S) or long (EN-L) isoforms or with control vector (Ctr). Cells were seeded on matrigel and exposed to hypoxia (1% oxygen, Hx) for 6 h or remained under normoxia (Ctr). (B) HMEC-1 cells were transfected with siRNA against β3-endonexin (siEN) or scrambled RNA (siCtr) and, in addition, transfected with an expression vector coding for I κ Bdn or with control vector or with siRNA against HIF-1 α (siHIF-1 α ). Cells were seeded on matrigel and exposed to hypoxia as described earlier. (C) For quantification of tube formation assays, total tube lengths were evaluated. Normoxic controls were set equal to 1 ( n =3, * p
Figure Legend Snippet: β3-endonexin decreases hypoxic tube formation. (A) HMEC-1 cells were transfected with expression vectors coding for either β3-endonexin short (EN-S) or long (EN-L) isoforms or with control vector (Ctr). Cells were seeded on matrigel and exposed to hypoxia (1% oxygen, Hx) for 6 h or remained under normoxia (Ctr). (B) HMEC-1 cells were transfected with siRNA against β3-endonexin (siEN) or scrambled RNA (siCtr) and, in addition, transfected with an expression vector coding for I κ Bdn or with control vector or with siRNA against HIF-1 α (siHIF-1 α ). Cells were seeded on matrigel and exposed to hypoxia as described earlier. (C) For quantification of tube formation assays, total tube lengths were evaluated. Normoxic controls were set equal to 1 ( n =3, * p

Techniques Used: Transfection, Expressing, Plasmid Preparation

β3-endonexin deficiency promotes the angiogenic response in vivo . (A) HMEC-1 cells were transfected with an expression vector for β3-endonexin long (EN-L) or control vector (Ctr), seeded on matrigel, and exposed to VEGF (1 μg/μl) for 6 h or remained untreated (Ctr). A representative figure is shown ( n =3). (B) HMEC-1 cells transfected with RNAi against β3-endonexin (siEN) or with scrambled RNA (siCtr) were mixed with matrigel. Plugs were injected subcutaneously in mice and excised after 7 days. Immunohistochemistry was performed with an antibody against CD31 to stain for human endothelial cells. Representative figures are shown ( n =3, * p
Figure Legend Snippet: β3-endonexin deficiency promotes the angiogenic response in vivo . (A) HMEC-1 cells were transfected with an expression vector for β3-endonexin long (EN-L) or control vector (Ctr), seeded on matrigel, and exposed to VEGF (1 μg/μl) for 6 h or remained untreated (Ctr). A representative figure is shown ( n =3). (B) HMEC-1 cells transfected with RNAi against β3-endonexin (siEN) or with scrambled RNA (siCtr) were mixed with matrigel. Plugs were injected subcutaneously in mice and excised after 7 days. Immunohistochemistry was performed with an antibody against CD31 to stain for human endothelial cells. Representative figures are shown ( n =3, * p

Techniques Used: In Vivo, Transfection, Expressing, Plasmid Preparation, Injection, Mouse Assay, Immunohistochemistry, Staining

β3-endonexin decreases HIF activity and HIF-1α protein levels under hypoxia. (A) HMEC-1 cells were co-transfected with a luciferase construct driven by three hypoxia-response elements (HRE) from the erythropoietin gene (EPO-HRE) and expression vectors coding for either β3-endonexin short (EN-S) or long (EN-L) isoforms or with control vector (Ctr) or with RNAi against β3-endonexin (siEN) or with scrambled RNA (siCtr). HMEC-1 cells were exposed to hypoxia (1% oxygen, Hx) for 8 h, and luciferase assay was performed. Normoxic controls (Ctr) were set equal to 100% ( n =3, * p
Figure Legend Snippet: β3-endonexin decreases HIF activity and HIF-1α protein levels under hypoxia. (A) HMEC-1 cells were co-transfected with a luciferase construct driven by three hypoxia-response elements (HRE) from the erythropoietin gene (EPO-HRE) and expression vectors coding for either β3-endonexin short (EN-S) or long (EN-L) isoforms or with control vector (Ctr) or with RNAi against β3-endonexin (siEN) or with scrambled RNA (siCtr). HMEC-1 cells were exposed to hypoxia (1% oxygen, Hx) for 8 h, and luciferase assay was performed. Normoxic controls (Ctr) were set equal to 100% ( n =3, * p

Techniques Used: Activity Assay, Transfection, Luciferase, Construct, Expressing, Plasmid Preparation

β3-endonexin decreases mRNA levels of HIF-1α and its target genes under hypoxia. (A–C) HMEC-1 cells were transfected with RNAi against β3-endonexin (siEN) or with scrambled RNA (siCtr) and exposed to hypoxia for 1, 4, and 8 h or cultivated under normoxia (0 h). RT-qPCR analyses were performed with primers amplifying human HIF-1 α (A) , VEGF (B), or GAPDH (C) as well as 18S rRNA for normalization. Control levels at normoxia (siCtr, 0 h) were set equal to 1 ( n =3, * p
Figure Legend Snippet: β3-endonexin decreases mRNA levels of HIF-1α and its target genes under hypoxia. (A–C) HMEC-1 cells were transfected with RNAi against β3-endonexin (siEN) or with scrambled RNA (siCtr) and exposed to hypoxia for 1, 4, and 8 h or cultivated under normoxia (0 h). RT-qPCR analyses were performed with primers amplifying human HIF-1 α (A) , VEGF (B), or GAPDH (C) as well as 18S rRNA for normalization. Control levels at normoxia (siCtr, 0 h) were set equal to 1 ( n =3, * p

Techniques Used: Transfection, Quantitative RT-PCR

β3-endonexin diminishes endothelial proliferative responses under hypoxia. HMEC-1 cells were transfected with expression vectors coding for either β3-endonexin short (EN-S) or long (EN-L) isoform or with control vector (Ctr), or with siRNA against β3-endonexin (siEN) or scrambled RNA (siCtr). Alternatively, HMEC-1 cells were transfected with siRNA against β3-endonexin (siEN) or scrambled RNA (siCtr) and, in addition, transfected with an expression vector coding for I κ Bdn or with control vector (Ctr) or with siRNA against HIF-1 α (siHIF-1 α ). Cells were exposed to hypoxia (1% oxygen, Hx) for 24 h or remained under normoxia (Ctr), and BrdU incorporation assay was performed. Normoxic controls were set equal to 100% ( n =3, * p
Figure Legend Snippet: β3-endonexin diminishes endothelial proliferative responses under hypoxia. HMEC-1 cells were transfected with expression vectors coding for either β3-endonexin short (EN-S) or long (EN-L) isoform or with control vector (Ctr), or with siRNA against β3-endonexin (siEN) or scrambled RNA (siCtr). Alternatively, HMEC-1 cells were transfected with siRNA against β3-endonexin (siEN) or scrambled RNA (siCtr) and, in addition, transfected with an expression vector coding for I κ Bdn or with control vector (Ctr) or with siRNA against HIF-1 α (siHIF-1 α ). Cells were exposed to hypoxia (1% oxygen, Hx) for 24 h or remained under normoxia (Ctr), and BrdU incorporation assay was performed. Normoxic controls were set equal to 100% ( n =3, * p

Techniques Used: Transfection, Expressing, Plasmid Preparation, BrdU Incorporation Assay

5) Product Images from "Dexamethasone Enhances ATP-Induced Inflammatory Responses in Endothelial Cells"

Article Title: Dexamethasone Enhances ATP-Induced Inflammatory Responses in Endothelial Cells

Journal: The Journal of Pharmacology and Experimental Therapeutics

doi: 10.1124/jpet.110.171975

Dexamethasone up-regulates the P2Y 2 R expression in HMEC-1 cells. A, cells cultured with 100 nM dexamethasone for 2, 4, 6, 12, 24, 36, and 48 h increased P2Y 2 R mRNA, with a maximal response by 6 h that remained elevated for 48 h. B, cells were incubated
Figure Legend Snippet: Dexamethasone up-regulates the P2Y 2 R expression in HMEC-1 cells. A, cells cultured with 100 nM dexamethasone for 2, 4, 6, 12, 24, 36, and 48 h increased P2Y 2 R mRNA, with a maximal response by 6 h that remained elevated for 48 h. B, cells were incubated

Techniques Used: Expressing, Cell Culture, Incubation

Effect of dexamethasone on [Ca 2+ ] i in response to ATP. HMEC-1 cells were cultured in the presence of DEX at concentrations of 10, 100, or 1000 nM for 36 h, followed by the addition of 100 μM ATP, and then [Ca 2+ ] i was measured immediately. The
Figure Legend Snippet: Effect of dexamethasone on [Ca 2+ ] i in response to ATP. HMEC-1 cells were cultured in the presence of DEX at concentrations of 10, 100, or 1000 nM for 36 h, followed by the addition of 100 μM ATP, and then [Ca 2+ ] i was measured immediately. The

Techniques Used: Cell Culture

Time and dose effects of ATP and dexamethasone on ATP-induced IL-6 release by HMEC-1 cells. A, HMEC-1 cells were incubated in depleted medium with ATP and vehicle for 1, 2, 6, 12, or 24 h. ATP increased IL-6 release after 24 h. B, dexamethasone increased
Figure Legend Snippet: Time and dose effects of ATP and dexamethasone on ATP-induced IL-6 release by HMEC-1 cells. A, HMEC-1 cells were incubated in depleted medium with ATP and vehicle for 1, 2, 6, 12, or 24 h. ATP increased IL-6 release after 24 h. B, dexamethasone increased

Techniques Used: Incubation

Effect of dexamethasone on ATP-induced IL-6 release by HMEC-1 cells. HMEC-1 cells were cultured with or without the presence of dexamethasone (DEX; 100 nM) for 36 h, followed by the addition of vehicle, LPS (10 ng/ml), ATP (100 μM), or UTP (100
Figure Legend Snippet: Effect of dexamethasone on ATP-induced IL-6 release by HMEC-1 cells. HMEC-1 cells were cultured with or without the presence of dexamethasone (DEX; 100 nM) for 36 h, followed by the addition of vehicle, LPS (10 ng/ml), ATP (100 μM), or UTP (100

Techniques Used: Cell Culture

Effect of ATP on IL-6 mRNA induction by HMEC-1 cells. A, HMEC-1 cells were cultured with or without 100 μM ATP for 5, 15, 30, 60, 120, 240, and 360 min, and mRNA expressions were measured by real-time RT-PCR. The data are shown as fold change
Figure Legend Snippet: Effect of ATP on IL-6 mRNA induction by HMEC-1 cells. A, HMEC-1 cells were cultured with or without 100 μM ATP for 5, 15, 30, 60, 120, 240, and 360 min, and mRNA expressions were measured by real-time RT-PCR. The data are shown as fold change

Techniques Used: Cell Culture, Quantitative RT-PCR

Dexamethasone enhancement of ATP-induced IL-6 is glucocorticoid receptor-dependent and requires P2Y 2 R and downstream activation of PLC and p38 MAPK. A, HMEC-1 cells were cultured in vehicle and mifepristone (RU486; 10 μM) 30 min before the addition
Figure Legend Snippet: Dexamethasone enhancement of ATP-induced IL-6 is glucocorticoid receptor-dependent and requires P2Y 2 R and downstream activation of PLC and p38 MAPK. A, HMEC-1 cells were cultured in vehicle and mifepristone (RU486; 10 μM) 30 min before the addition

Techniques Used: Activation Assay, Planar Chromatography, Cell Culture

6) Product Images from "Calcitonin Gene-related Peptide Inhibits Chemokine Production by Human Dermal Microvascular Endothelial Cells"

Article Title: Calcitonin Gene-related Peptide Inhibits Chemokine Production by Human Dermal Microvascular Endothelial Cells

Journal: Brain, behavior, and immunity

doi: 10.1016/j.bbi.2011.02.007

CGRP prevents NF-κB binding to the promoters of CXCL1, CXCL8 and CCL2 and LPS-induced IκBα degradation in HMEC-1 cells. (A) CGRP inhibits NF-κB DNA binding to promoters. Nuclear extracts were prepared from HMEC-1 cells
Figure Legend Snippet: CGRP prevents NF-κB binding to the promoters of CXCL1, CXCL8 and CCL2 and LPS-induced IκBα degradation in HMEC-1 cells. (A) CGRP inhibits NF-κB DNA binding to promoters. Nuclear extracts were prepared from HMEC-1 cells

Techniques Used: Binding Assay

HMEC-1 cells (A) and pHMDECs (B) express mRNA for components of the AM and CGRP receptors (RAMP1/CL, RAMP2/CL and RAMP3/CL). Total RNA was extracted from unstimulated HMEC-1 cells and subjected to RT-PCR with primers specific for RAMP1, RAMP2, RAMP3,
Figure Legend Snippet: HMEC-1 cells (A) and pHMDECs (B) express mRNA for components of the AM and CGRP receptors (RAMP1/CL, RAMP2/CL and RAMP3/CL). Total RNA was extracted from unstimulated HMEC-1 cells and subjected to RT-PCR with primers specific for RAMP1, RAMP2, RAMP3,

Techniques Used: Reverse Transcription Polymerase Chain Reaction

CGRP inhibits LPS-induced CXCL8, CCL2, and CXCL1 production by HMEC-1 cells. (A) Time-dependent chemokine inhibition by CGRP. HMEC-1 cells (0.25 ×10 6 cells/ml) were exposed to LPS in the presence or absence of CGRP, and supernatants were collected
Figure Legend Snippet: CGRP inhibits LPS-induced CXCL8, CCL2, and CXCL1 production by HMEC-1 cells. (A) Time-dependent chemokine inhibition by CGRP. HMEC-1 cells (0.25 ×10 6 cells/ml) were exposed to LPS in the presence or absence of CGRP, and supernatants were collected

Techniques Used: Inhibition

CGRP inhibits the expression of LPS-induced chemokines at the mRNA level. HMEC-1 cells (1.5 ×10 6 cells) were stimulated with LPS (1 μg/ml) in the presence or absence of CGRP (10 nM) for 12 h. Total RNA was extracted and transferred onto
Figure Legend Snippet: CGRP inhibits the expression of LPS-induced chemokines at the mRNA level. HMEC-1 cells (1.5 ×10 6 cells) were stimulated with LPS (1 μg/ml) in the presence or absence of CGRP (10 nM) for 12 h. Total RNA was extracted and transferred onto

Techniques Used: Expressing

Exposure of HMEC-1 cells to CGRP during stimulation inhibits chemotaxis of neutrophils and monocunclear cells towards LPS-stimulated HMEC-1 cells or supernatants conditioned by LPS-stimulated HMEC-1 cells. (A) Inhibition of chemotaxis towards stimulated
Figure Legend Snippet: Exposure of HMEC-1 cells to CGRP during stimulation inhibits chemotaxis of neutrophils and monocunclear cells towards LPS-stimulated HMEC-1 cells or supernatants conditioned by LPS-stimulated HMEC-1 cells. (A) Inhibition of chemotaxis towards stimulated

Techniques Used: Chemotaxis Assay, Inhibition

CGRP 8-37 and BIBN4096BS prevent CGRP-induced suppression of CXCL8, CCL2, and CXCL1 expression. (A) CGRP 8-37 . HMEC-1 cells were stimulated with 10 nM of CGRP in the presence or absence of various concentrations (0–1000 nM) of the CGRP antagonist
Figure Legend Snippet: CGRP 8-37 and BIBN4096BS prevent CGRP-induced suppression of CXCL8, CCL2, and CXCL1 expression. (A) CGRP 8-37 . HMEC-1 cells were stimulated with 10 nM of CGRP in the presence or absence of various concentrations (0–1000 nM) of the CGRP antagonist

Techniques Used: Expressing

7) Product Images from "A long-term "memory" of HIF induction in response to chronic mild decreased oxygen after oxygen normalization"

Article Title: A long-term "memory" of HIF induction in response to chronic mild decreased oxygen after oxygen normalization

Journal: BMC Cardiovascular Disorders

doi: 10.1186/1471-2261-7-4

Effect of chronic relative hypoxia on a marker of cell proliferation in HMEC-1 cells . HMEC-1 cells were exposed to normoxia (20% O2) or to various levels of relative hypoxia for 3 weeks followed by a one hour incubation with 5 μM CFDA-AM and fluorescence measurement as in methods. The data represent the mean +/- SEM of triplicate wells from 3 independent experiments. ** = P
Figure Legend Snippet: Effect of chronic relative hypoxia on a marker of cell proliferation in HMEC-1 cells . HMEC-1 cells were exposed to normoxia (20% O2) or to various levels of relative hypoxia for 3 weeks followed by a one hour incubation with 5 μM CFDA-AM and fluorescence measurement as in methods. The data represent the mean +/- SEM of triplicate wells from 3 independent experiments. ** = P

Techniques Used: Marker, Incubation, Fluorescence

Effect of adenoviral siRNA on HIF-1α protein (A) and cell proliferation (B) in HMEC-1 cells . Adenoviral vectors containing hairpin siRNAs against HIF-1α were created as in the methods. Panel A. HMEC-1 cells were infected with 50–100 PFU/cell adenovirus or 5 μM YC-1, exposed to 5% O 2 for 48 hours then cell lysates made and HIF-1α protein expression assessed by Western blot. Representative bands for HIF-1α are shown. Panel B. HMEC-1 cells were infected with adenovirus or treated with 5 μM YC-1 every 3 days for 7 days and cell proliferation assessed using the CFDA-AM assay as in methods. The data represent the mean +/- SEM of triplicate wells from 3 independent experiments. *** = P
Figure Legend Snippet: Effect of adenoviral siRNA on HIF-1α protein (A) and cell proliferation (B) in HMEC-1 cells . Adenoviral vectors containing hairpin siRNAs against HIF-1α were created as in the methods. Panel A. HMEC-1 cells were infected with 50–100 PFU/cell adenovirus or 5 μM YC-1, exposed to 5% O 2 for 48 hours then cell lysates made and HIF-1α protein expression assessed by Western blot. Representative bands for HIF-1α are shown. Panel B. HMEC-1 cells were infected with adenovirus or treated with 5 μM YC-1 every 3 days for 7 days and cell proliferation assessed using the CFDA-AM assay as in methods. The data represent the mean +/- SEM of triplicate wells from 3 independent experiments. *** = P

Techniques Used: Infection, Expressing, Western Blot

8) Product Images from "Adenosine A2B receptor stimulates angiogenesis by inducing VEGF and eNOS in human microvascular endothelial cells"

Article Title: Adenosine A2B receptor stimulates angiogenesis by inducing VEGF and eNOS in human microvascular endothelial cells

Journal: Experimental Biology and Medicine

doi: 10.1177/1535370215584939

Effects of adenosine and NECA on the tube formation of HMEC-1. HMEC-1 cells were seeded in a 96-well plate pre-coated with Matrigel and were stimulated with a series of A 2B receptor agonist adenosine and NECA for 24 h. Representative light microscopy
Figure Legend Snippet: Effects of adenosine and NECA on the tube formation of HMEC-1. HMEC-1 cells were seeded in a 96-well plate pre-coated with Matrigel and were stimulated with a series of A 2B receptor agonist adenosine and NECA for 24 h. Representative light microscopy

Techniques Used: Light Microscopy

cAMP-PKA-CREB pathway is account for the upregulation of VEGF. (A) Accumulation of cAMP induced by the activator Forskolin, NECA or MRS 1754, alone and in combination, in HMEC-1 cells and A 2B receptor siRNA transfected HMEC-1. (B) Western blotting analysis
Figure Legend Snippet: cAMP-PKA-CREB pathway is account for the upregulation of VEGF. (A) Accumulation of cAMP induced by the activator Forskolin, NECA or MRS 1754, alone and in combination, in HMEC-1 cells and A 2B receptor siRNA transfected HMEC-1. (B) Western blotting analysis

Techniques Used: Transfection, Western Blot

9) Product Images from "ExoU Activates NF-?B and Increases IL-8/KC Secretion during Pseudomonas aeruginosa Infection"

Article Title: ExoU Activates NF-?B and Increases IL-8/KC Secretion during Pseudomonas aeruginosa Infection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0041772

ExoU activates p65/p50 NF-κB and increases IL-8 expression and secretion in HMEC-1 capillary endothelial cells. In (A), representative agarose gels of three different semi-quantitative RT-PCR assays carried out in duplicate. In (B), graph shows the means ± SEM of values obtained in three Real Time qRT-PCR assays. **p
Figure Legend Snippet: ExoU activates p65/p50 NF-κB and increases IL-8 expression and secretion in HMEC-1 capillary endothelial cells. In (A), representative agarose gels of three different semi-quantitative RT-PCR assays carried out in duplicate. In (B), graph shows the means ± SEM of values obtained in three Real Time qRT-PCR assays. **p

Techniques Used: Expressing, Quantitative RT-PCR

10) Product Images from "Differentially Expressed Genes of Human Microvascular Endothelial Cells in Response to Anti-Dengue Virus NS1 Antibodies by Suppression Subtractive Hybridization"

Article Title: Differentially Expressed Genes of Human Microvascular Endothelial Cells in Response to Anti-Dengue Virus NS1 Antibodies by Suppression Subtractive Hybridization

Journal: Viral Immunology

doi: 10.1089/vim.2012.0063

Relative fold expression of five different target genes, nuclear ubiquitous casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1), CD59, S100A6, aryl hydrocarbon receptor nuclear translocator isoform 1/3 (ARNT1/3), and ADP-ribosylation factor-like 6 interacting protein (ARL6IP), expressed in the HMEC-1 cells incubated with anti-NS1 Abs ( solid bar ) compared to normal rabbit IgG incubated cells ( open bar ) and standardized against β-actin as the internal reference, were analyzed by real-time RT-PCR at 2, 4, 6, 8, 10, 12, 24, and 48 hours post incubation. Samples from each time point were tested in triplicate and the data represent the mean (±1 standard deviation) relative expression level was used for analysis that the target gene is downregulated, the same, or upregulated, respectively, compared with the control (* p
Figure Legend Snippet: Relative fold expression of five different target genes, nuclear ubiquitous casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1), CD59, S100A6, aryl hydrocarbon receptor nuclear translocator isoform 1/3 (ARNT1/3), and ADP-ribosylation factor-like 6 interacting protein (ARL6IP), expressed in the HMEC-1 cells incubated with anti-NS1 Abs ( solid bar ) compared to normal rabbit IgG incubated cells ( open bar ) and standardized against β-actin as the internal reference, were analyzed by real-time RT-PCR at 2, 4, 6, 8, 10, 12, 24, and 48 hours post incubation. Samples from each time point were tested in triplicate and the data represent the mean (±1 standard deviation) relative expression level was used for analysis that the target gene is downregulated, the same, or upregulated, respectively, compared with the control (* p

Techniques Used: Expressing, Incubation, Quantitative RT-PCR, Standard Deviation

Related Articles

Transfection:

Article Title: Sustained Expression of Homeobox D10 Inhibits Angiogenesis
Article Snippet: .. HMEC-1 cells were transfected using Effectene (Qiagen, Valencia, CA) and pools of stable transfectants selected with 35 μg/ml of G418 (Life Technologies, Gaithersburg, MD). .. Total RNA isolation and Northern blot analysis was performed as previously described.

Article Title: Role of Delta-like 4 in Jagged1-induced tumour angiogenesis and tumour growth
Article Snippet: .. HMEC-1 cells were transfected with siRNA (20nM for siDLL4 and 5nM for siJAG1) using HiPerfect (QIAGEN) according to manufacturer's instructions. .. Hanging drop assay HMEC-1 cells were suspended in medium containing 0.2% (w/v) carboxymethylcellulose and seeded in non-adherent 60-microwell minitrays (Sigma).

Synthesized:

Article Title: The ?3-Integrin Binding Protein ?3-Endonexin Is a Novel Negative Regulator of Hypoxia-Inducible Factor-1
Article Snippet: .. Total RNA was isolated from HMEC-1 cells or from murine lung tissue using RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. cDNA was synthesized from isolated RNA using high-capacity cDNA reverse transcription kit (Qiagen) according to the manufacturer's instructions in a 6000 Rotor Gene Real-Time PCR System (Qiagen). .. RT-qPCR was performed using human gene-specific primers: ITGB3BP (β3 - endonexin consensus sequence) 5′-TCT CCA ACA ACT GGA ACT TGT C-3′(sense), 5′-TCC ATT TCT GTG CTT TTG CTC-3′ (anti-sense); HIF1A 5′-GAA GAC ATC GCG GGG AC-3′ (sense), 5′-TGG CTG CAT CTC GAG ACT TT-3′ (anti-sense); GAPDH 5′ - GTG AAC ATG AGA AGT ATG ACA AC-3′ (sense), 5′-CAT GAG TCC TTC CAC GAT ACC-3′ (anti-sense); VEGFA 5′-AGG AGG AGG GCA GAA TCA TCA-3′ (sense) , 5 ′-CTC GAT TGG ATG GCA AGT AGC T-3′ (anti-sense) ; 18S rRNA 5′-GTA ACC CGT TGA ACC CCA TT-3′ (sense), 5′-CCATCCAATCGGTAGTAGCG-3′ (anti-sense).

Isolation:

Article Title: The ?3-Integrin Binding Protein ?3-Endonexin Is a Novel Negative Regulator of Hypoxia-Inducible Factor-1
Article Snippet: .. Total RNA was isolated from HMEC-1 cells or from murine lung tissue using RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. cDNA was synthesized from isolated RNA using high-capacity cDNA reverse transcription kit (Qiagen) according to the manufacturer's instructions in a 6000 Rotor Gene Real-Time PCR System (Qiagen). .. RT-qPCR was performed using human gene-specific primers: ITGB3BP (β3 - endonexin consensus sequence) 5′-TCT CCA ACA ACT GGA ACT TGT C-3′(sense), 5′-TCC ATT TCT GTG CTT TTG CTC-3′ (anti-sense); HIF1A 5′-GAA GAC ATC GCG GGG AC-3′ (sense), 5′-TGG CTG CAT CTC GAG ACT TT-3′ (anti-sense); GAPDH 5′ - GTG AAC ATG AGA AGT ATG ACA AC-3′ (sense), 5′-CAT GAG TCC TTC CAC GAT ACC-3′ (anti-sense); VEGFA 5′-AGG AGG AGG GCA GAA TCA TCA-3′ (sense) , 5 ′-CTC GAT TGG ATG GCA AGT AGC T-3′ (anti-sense) ; 18S rRNA 5′-GTA ACC CGT TGA ACC CCA TT-3′ (sense), 5′-CCATCCAATCGGTAGTAGCG-3′ (anti-sense).

Article Title: Adenosine A2B receptor stimulates angiogenesis by inducing VEGF and eNOS in human microvascular endothelial cells
Article Snippet: .. HMEC-1 cells were separately incubated with 10 µmol/L CPA (A2A AR selective agonist), 10 µmol/L NECA, and 10 µmol/L MRS1754 (A2B AR inhibitor) for 24 h. Total RNA was isolated and purified using an RNeasy Plus Universal Kit, according to the manufacturer’s instructions (Qiagen, Santa Clara, CA, USA). .. RNA (1.5 µg) was reverse transcribed into cDNA that was utilized as a template in the subsequent RT-PCR amplifications using a SYBR Green Real-Time PCR Master Mix kit (Takara Biotechnology, Dalian, China).

RNA Extraction:

Article Title: A long-term "memory" of HIF induction in response to chronic mild decreased oxygen after oxygen normalization
Article Snippet: .. RNA Extraction and Quantitative Real-Time RT-PCR Total RNA was extracted from the control and treated HMEC-1 cells using the RNeasy kit (QIAGEN) immediately following the treatments. .. The concentration and purity of RNA samples were determined spectrophotometrically by absorbance at 260 and 280 nm, and the integrity was confirmed by agarose gel electrophoresis.

Article Title: Calcitonin Gene-related Peptide Inhibits Chemokine Production by Human Dermal Microvascular Endothelial Cells
Article Snippet: .. Total RNA was extracted from HMEC-1 cells and pHDMECs using a total RNA extraction kit as per the manufacturer’s instructions (Qiagen, Valencia, CA, USA). ..

Quantitative RT-PCR:

Article Title: A long-term "memory" of HIF induction in response to chronic mild decreased oxygen after oxygen normalization
Article Snippet: .. RNA Extraction and Quantitative Real-Time RT-PCR Total RNA was extracted from the control and treated HMEC-1 cells using the RNeasy kit (QIAGEN) immediately following the treatments. .. The concentration and purity of RNA samples were determined spectrophotometrically by absorbance at 260 and 280 nm, and the integrity was confirmed by agarose gel electrophoresis.

Purification:

Article Title: Adenosine A2B receptor stimulates angiogenesis by inducing VEGF and eNOS in human microvascular endothelial cells
Article Snippet: .. HMEC-1 cells were separately incubated with 10 µmol/L CPA (A2A AR selective agonist), 10 µmol/L NECA, and 10 µmol/L MRS1754 (A2B AR inhibitor) for 24 h. Total RNA was isolated and purified using an RNeasy Plus Universal Kit, according to the manufacturer’s instructions (Qiagen, Santa Clara, CA, USA). .. RNA (1.5 µg) was reverse transcribed into cDNA that was utilized as a template in the subsequent RT-PCR amplifications using a SYBR Green Real-Time PCR Master Mix kit (Takara Biotechnology, Dalian, China).

Real-time Polymerase Chain Reaction:

Article Title: The ?3-Integrin Binding Protein ?3-Endonexin Is a Novel Negative Regulator of Hypoxia-Inducible Factor-1
Article Snippet: .. Total RNA was isolated from HMEC-1 cells or from murine lung tissue using RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. cDNA was synthesized from isolated RNA using high-capacity cDNA reverse transcription kit (Qiagen) according to the manufacturer's instructions in a 6000 Rotor Gene Real-Time PCR System (Qiagen). .. RT-qPCR was performed using human gene-specific primers: ITGB3BP (β3 - endonexin consensus sequence) 5′-TCT CCA ACA ACT GGA ACT TGT C-3′(sense), 5′-TCC ATT TCT GTG CTT TTG CTC-3′ (anti-sense); HIF1A 5′-GAA GAC ATC GCG GGG AC-3′ (sense), 5′-TGG CTG CAT CTC GAG ACT TT-3′ (anti-sense); GAPDH 5′ - GTG AAC ATG AGA AGT ATG ACA AC-3′ (sense), 5′-CAT GAG TCC TTC CAC GAT ACC-3′ (anti-sense); VEGFA 5′-AGG AGG AGG GCA GAA TCA TCA-3′ (sense) , 5 ′-CTC GAT TGG ATG GCA AGT AGC T-3′ (anti-sense) ; 18S rRNA 5′-GTA ACC CGT TGA ACC CCA TT-3′ (sense), 5′-CCATCCAATCGGTAGTAGCG-3′ (anti-sense).

Incubation:

Article Title: Adenosine A2B receptor stimulates angiogenesis by inducing VEGF and eNOS in human microvascular endothelial cells
Article Snippet: .. HMEC-1 cells were separately incubated with 10 µmol/L CPA (A2A AR selective agonist), 10 µmol/L NECA, and 10 µmol/L MRS1754 (A2B AR inhibitor) for 24 h. Total RNA was isolated and purified using an RNeasy Plus Universal Kit, according to the manufacturer’s instructions (Qiagen, Santa Clara, CA, USA). .. RNA (1.5 µg) was reverse transcribed into cDNA that was utilized as a template in the subsequent RT-PCR amplifications using a SYBR Green Real-Time PCR Master Mix kit (Takara Biotechnology, Dalian, China).

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  • 92
    Qiagen hmec 1 cells
    DLL4 and JAG1 induced endogenous JAG1 expression in tumour tissues A . Expression of JAG1 (red) and CD31 (green) as revealed by immunofluorescence double staining of tumour sections of U87. Magnification 200X. B . Endogenous hJAG1 expression in parental U87 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental U87 cells. hJAG1 expression in GFP-negative U87 cells was detected by FACS staining with anti-human JAG1 mAb (64D). ANOVA with Bonferroni's post-test. N = 3, Error bars represent SD. C . Endogenous hJAG1 expression in parental <t>HMEC-1</t> cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental HMEC-1 cells. hJAG1 expression in GFP-negative HMEC-1 cells was detected by FACS staining with anti-human JAG1 mAb (64D). ANOVA with Bonferroni's post-test. NS, no statistical difference. N = 3, Error bars represent SD. * P
    Hmec 1 Cells, supplied by Qiagen, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen hmec 1 myocardial cells
    curcumin (Cur) impairs endothelin-1 (ET-1) production in Trypanosoma cruzi -infected human microvascular endothelial cells <t>(HMEC-1)</t> through downregulation of Ca 2+ -sensitive NFAT signalling. The involvement of Ca 2+ -dependent mechanisms in T. cruzi -promoted ET-1 release from infected HMEC-1 was verified. (A) Soluble ET-1 levels in the media of uninfected (PBS) or infected ( T. cruzi ) cells, cultured in the presence or in the absence of Cur at different concentrations (13.5 and 27.0 μM), were measured by enzyme-linked immunosorbent assay (ELISA). In some experiments, the cells were pre-treated for 1 h with chemical inhibitors of NFAT or c-Src kinase-mediated pathways (5 μM CsA or 20 μM Src-I1, respectively), and their effect on ET-1 secretion was further determined. Data are the means ± standard deviations of three independent experiments, each performed in triplicate. *p
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    Cluster analysis of the gene expression. Clustering of differentially expressed genes in the <t>HMEC-1</t> cells infected with LV-Con or <t>LV-shGPR4.</t>
    Lv Shgpr4 Infected Hmec 1 Cells, supplied by Qiagen, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DLL4 and JAG1 induced endogenous JAG1 expression in tumour tissues A . Expression of JAG1 (red) and CD31 (green) as revealed by immunofluorescence double staining of tumour sections of U87. Magnification 200X. B . Endogenous hJAG1 expression in parental U87 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental U87 cells. hJAG1 expression in GFP-negative U87 cells was detected by FACS staining with anti-human JAG1 mAb (64D). ANOVA with Bonferroni's post-test. N = 3, Error bars represent SD. C . Endogenous hJAG1 expression in parental HMEC-1 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental HMEC-1 cells. hJAG1 expression in GFP-negative HMEC-1 cells was detected by FACS staining with anti-human JAG1 mAb (64D). ANOVA with Bonferroni's post-test. NS, no statistical difference. N = 3, Error bars represent SD. * P

    Journal: Oncotarget

    Article Title: Role of Delta-like 4 in Jagged1-induced tumour angiogenesis and tumour growth

    doi: 10.18632/oncotarget.16969

    Figure Lengend Snippet: DLL4 and JAG1 induced endogenous JAG1 expression in tumour tissues A . Expression of JAG1 (red) and CD31 (green) as revealed by immunofluorescence double staining of tumour sections of U87. Magnification 200X. B . Endogenous hJAG1 expression in parental U87 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental U87 cells. hJAG1 expression in GFP-negative U87 cells was detected by FACS staining with anti-human JAG1 mAb (64D). ANOVA with Bonferroni's post-test. N = 3, Error bars represent SD. C . Endogenous hJAG1 expression in parental HMEC-1 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental HMEC-1 cells. hJAG1 expression in GFP-negative HMEC-1 cells was detected by FACS staining with anti-human JAG1 mAb (64D). ANOVA with Bonferroni's post-test. NS, no statistical difference. N = 3, Error bars represent SD. * P

    Article Snippet: HMEC-1 cells were transfected with siRNA (20nM for siDLL4 and 5nM for siJAG1) using HiPerfect (QIAGEN) according to manufacturer's instructions.

    Techniques: Expressing, Immunofluorescence, Double Staining, Co-Culture Assay, FACS, Staining

    DLL4 and JAG1 induced endogenous DLL4 expression in tumour vessels A . Expression of DLL4 (red) and CD31 (green) as revealed by immunofluorescence double staining of tumour sections of U87. Magnification 200X. B . Endogenous hDLL4 expression in parental U87 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental U87 cells. hDLL4 expression in GFP-negative U87 cells was detected by FACS staining with anti-DLL4 antibody (recognised both human and mouse DLL4). ANOVA with Bonferroni's post-test. NS, no statistical difference. N = 3. Error bars represent SD. C . Endogenous hDLL4 expression in parental HMEC-1 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental HMEC-1 cells. hDLL4 expression in GFP-negative HMEC-1 cells was detected by FACS staining with anti-DLL4 antibody. ANOVA with Bonferroni's post-test. NS, no statistical difference. N = 3, Error bars represent SD. * P

    Journal: Oncotarget

    Article Title: Role of Delta-like 4 in Jagged1-induced tumour angiogenesis and tumour growth

    doi: 10.18632/oncotarget.16969

    Figure Lengend Snippet: DLL4 and JAG1 induced endogenous DLL4 expression in tumour vessels A . Expression of DLL4 (red) and CD31 (green) as revealed by immunofluorescence double staining of tumour sections of U87. Magnification 200X. B . Endogenous hDLL4 expression in parental U87 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental U87 cells. hDLL4 expression in GFP-negative U87 cells was detected by FACS staining with anti-DLL4 antibody (recognised both human and mouse DLL4). ANOVA with Bonferroni's post-test. NS, no statistical difference. N = 3. Error bars represent SD. C . Endogenous hDLL4 expression in parental HMEC-1 cells (GFP-negative) sorted from co-culture of parental U87, U87-EV, U87-mDLL4 or U87-mJAG1 (GFP-positive) with an equal amount of parental HMEC-1 cells. hDLL4 expression in GFP-negative HMEC-1 cells was detected by FACS staining with anti-DLL4 antibody. ANOVA with Bonferroni's post-test. NS, no statistical difference. N = 3, Error bars represent SD. * P

    Article Snippet: HMEC-1 cells were transfected with siRNA (20nM for siDLL4 and 5nM for siJAG1) using HiPerfect (QIAGEN) according to manufacturer's instructions.

    Techniques: Expressing, Immunofluorescence, Double Staining, Co-Culture Assay, FACS, Staining

    DLL4 and JAG1 activated Notch signalling and affected sprouting angiogenesis in vitro . A . Expression profile of Notch target genes in HMEC-1 stimulated with rhDLL4 or rrJAG1 over a time course. QPCR was used to determine mRNA levels of DLL4, JAG1, HEY1 and HEY2. Fold changes were obtained by normalizing against the EV control. B . Expression profile of Notch target genes in HMEC-1 over-expressing mDLL4 or mJAG1 by retrovirus transductions. QPCR was used to determine mRNA levels of DLL4, JAG1, HEY1 and HEY2 (ANOVA with Bonferroni's post-test). C . Expression of endogenous hDLL4 and hJAG1 proteins in parental HMEC-1 cells (GFP-negative) sorted from co-culture of EV-, mDLL4- or mJAG1-overexpressing HMEC-1 with an equal amount of parental HMEC-1 cells by FACS analysis (ANOVA with Bonferroni's post-test). D . Effect of mDLL4 and mJAG1 expressed in HMEC-1 cells on sprouting in HMEC-1 spheroids and treated with DBZ. Average lengths of three longest sprouts were calculated for statistical test (ANOVA with Bonferroni's post-test). Graphs are means of 3 independent experiments. E . Effect of knockdown of DLL4 in HMEC-1 cells by specific siDLL4 on sprouting in HMEC-1 spheroids. Average lengths of three longest sprouts were calculated for statistical test (ANOVA with Bonferroni's post-test). Graphs are means of 3 independent experiments. F . Effect of knockdown of JAG1 in HMEC-1 cells by specific siJAG1 on sprouting in HMEC-1 spheroids. Average lengths of three longest sprouts were calculated for statistical test (ANOVA with Bonferroni's post-test). Graphs are means of 3 independent experiments. * P

    Journal: Oncotarget

    Article Title: Role of Delta-like 4 in Jagged1-induced tumour angiogenesis and tumour growth

    doi: 10.18632/oncotarget.16969

    Figure Lengend Snippet: DLL4 and JAG1 activated Notch signalling and affected sprouting angiogenesis in vitro . A . Expression profile of Notch target genes in HMEC-1 stimulated with rhDLL4 or rrJAG1 over a time course. QPCR was used to determine mRNA levels of DLL4, JAG1, HEY1 and HEY2. Fold changes were obtained by normalizing against the EV control. B . Expression profile of Notch target genes in HMEC-1 over-expressing mDLL4 or mJAG1 by retrovirus transductions. QPCR was used to determine mRNA levels of DLL4, JAG1, HEY1 and HEY2 (ANOVA with Bonferroni's post-test). C . Expression of endogenous hDLL4 and hJAG1 proteins in parental HMEC-1 cells (GFP-negative) sorted from co-culture of EV-, mDLL4- or mJAG1-overexpressing HMEC-1 with an equal amount of parental HMEC-1 cells by FACS analysis (ANOVA with Bonferroni's post-test). D . Effect of mDLL4 and mJAG1 expressed in HMEC-1 cells on sprouting in HMEC-1 spheroids and treated with DBZ. Average lengths of three longest sprouts were calculated for statistical test (ANOVA with Bonferroni's post-test). Graphs are means of 3 independent experiments. E . Effect of knockdown of DLL4 in HMEC-1 cells by specific siDLL4 on sprouting in HMEC-1 spheroids. Average lengths of three longest sprouts were calculated for statistical test (ANOVA with Bonferroni's post-test). Graphs are means of 3 independent experiments. F . Effect of knockdown of JAG1 in HMEC-1 cells by specific siJAG1 on sprouting in HMEC-1 spheroids. Average lengths of three longest sprouts were calculated for statistical test (ANOVA with Bonferroni's post-test). Graphs are means of 3 independent experiments. * P

    Article Snippet: HMEC-1 cells were transfected with siRNA (20nM for siDLL4 and 5nM for siJAG1) using HiPerfect (QIAGEN) according to manufacturer's instructions.

    Techniques: In Vitro, Expressing, Real-time Polymerase Chain Reaction, Co-Culture Assay, FACS

    Surface vimentin is localized along the periphery of HMEC-1. (A–D) Cells were stained for vimentin using the rabbit anti-vimentin H-84 antibody. For negative controls, cells were stained with secondary anti-rabbit IgG antibody alone. Representative phase image of cells (left column), image of the nuclei (middle column), and H-84 anti-vimentin antibody fluorescence (right column) are shown for (A) permeabilized HMEC-1 strained for intracellular vimentin; (B) permeabilized HMEC-1 incubated with anti-rabbit IgG alone as negative controls; (C) nonpermeabilized HMEC-1 stained for surface vimentin; (D) nonpermeabilized HMEC-1 incubated with anti-rabbit IgG alone as negative controls. Scale bar shown in white is 20 μm. White arrows point at the localization of surface vimentin at cell–cell junctions.

    Journal: Molecular Biology of the Cell

    Article Title: Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin

    doi: 10.1091/mbc.E18-04-0228

    Figure Lengend Snippet: Surface vimentin is localized along the periphery of HMEC-1. (A–D) Cells were stained for vimentin using the rabbit anti-vimentin H-84 antibody. For negative controls, cells were stained with secondary anti-rabbit IgG antibody alone. Representative phase image of cells (left column), image of the nuclei (middle column), and H-84 anti-vimentin antibody fluorescence (right column) are shown for (A) permeabilized HMEC-1 strained for intracellular vimentin; (B) permeabilized HMEC-1 incubated with anti-rabbit IgG alone as negative controls; (C) nonpermeabilized HMEC-1 stained for surface vimentin; (D) nonpermeabilized HMEC-1 incubated with anti-rabbit IgG alone as negative controls. Scale bar shown in white is 20 μm. White arrows point at the localization of surface vimentin at cell–cell junctions.

    Article Snippet: HMEC-1 cells were treated with control or experimental siRNA as described above. mRNA was harvested using the RNeasy Micro Kit (Qiagen; 74004), and cDNA was prepared using the Superscript III First-strand Synthesis SuperMix (ThermoFisher; 18080–400).

    Techniques: Staining, Fluorescence, Incubation

    Infection of HMEC-1 by Lm is in part mediated by InlB in a manner independent of matrix stiffness. (A) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: wild type (circle); Δ inlA (square); Δ inlB (cross); Δ inlF (diamond; actAp::mTagRFP). The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. (B) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: Δ actA (black circles); Δ actA/ Δ inlB (gray squares; actAp::mTagRFP). The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. (C) Boxplots of percentage of HMEC-1 infected with Lm as a function of substrate stiffness ( N = 5–6 replicates). HMEC-1 were infected with the indicated Lm strains: Δ actA (gray); Δ actA/ Δ inlB (black; actAp::mTagRFP) at an MOI of 20. Infection was analyzed by flow cytometry 7–8 h after infection. Representative data come from one of three independent experiments. One or two asterisks denote statistically significant differences between the medians of two distributions (

    Journal: Molecular Biology of the Cell

    Article Title: Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin

    doi: 10.1091/mbc.E18-04-0228

    Figure Lengend Snippet: Infection of HMEC-1 by Lm is in part mediated by InlB in a manner independent of matrix stiffness. (A) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: wild type (circle); Δ inlA (square); Δ inlB (cross); Δ inlF (diamond; actAp::mTagRFP). The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. (B) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: Δ actA (black circles); Δ actA/ Δ inlB (gray squares; actAp::mTagRFP). The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. (C) Boxplots of percentage of HMEC-1 infected with Lm as a function of substrate stiffness ( N = 5–6 replicates). HMEC-1 were infected with the indicated Lm strains: Δ actA (gray); Δ actA/ Δ inlB (black; actAp::mTagRFP) at an MOI of 20. Infection was analyzed by flow cytometry 7–8 h after infection. Representative data come from one of three independent experiments. One or two asterisks denote statistically significant differences between the medians of two distributions (

    Article Snippet: HMEC-1 cells were treated with control or experimental siRNA as described above. mRNA was harvested using the RNeasy Micro Kit (Qiagen; 74004), and cDNA was prepared using the Superscript III First-strand Synthesis SuperMix (ThermoFisher; 18080–400).

    Techniques: Infection, Flow Cytometry, Cytometry

    Uptake of Lm by HMEC-1 depends on the stiffness of the matrix on which cells reside. HMEC-1 residing on PA hydrogels of varying stiffness coated with collagen I were infected with Δ actA Lm (actAp::mTagRFP). Infection was analyzed by flow cytometry 7–8 h postinfection. Bacteria were added at a multiplicity of infection (MOI) between 30 and 50 bacteria per host cell. (A–D) Histograms of the logarithm of bacterial fluorescence intensity per cell for HMEC-1 plated on 0.6-kPa (A), 3-kPa (B), 20-kPa (C), and 70-kPa (D) PA hydrogels. Histograms for N = 5 replicates are shown in different colors. The histogram of control uninfected cells is shown in purple. Based on the autofluorescence of the control group, a gate is defined (see black and red lines) showing what is considered uninfected (left, black line) and infected (right, red line). (E) Boxplots of percentage of HMEC-1 infected with Δ actA Lm vs. hydrogel stiffness for the data shown in panels A–D. Circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test; for details about boxplots see Materials and Methods ). One or two asterisks denote statistically significant differences between the medians of two distributions (

    Journal: Molecular Biology of the Cell

    Article Title: Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin

    doi: 10.1091/mbc.E18-04-0228

    Figure Lengend Snippet: Uptake of Lm by HMEC-1 depends on the stiffness of the matrix on which cells reside. HMEC-1 residing on PA hydrogels of varying stiffness coated with collagen I were infected with Δ actA Lm (actAp::mTagRFP). Infection was analyzed by flow cytometry 7–8 h postinfection. Bacteria were added at a multiplicity of infection (MOI) between 30 and 50 bacteria per host cell. (A–D) Histograms of the logarithm of bacterial fluorescence intensity per cell for HMEC-1 plated on 0.6-kPa (A), 3-kPa (B), 20-kPa (C), and 70-kPa (D) PA hydrogels. Histograms for N = 5 replicates are shown in different colors. The histogram of control uninfected cells is shown in purple. Based on the autofluorescence of the control group, a gate is defined (see black and red lines) showing what is considered uninfected (left, black line) and infected (right, red line). (E) Boxplots of percentage of HMEC-1 infected with Δ actA Lm vs. hydrogel stiffness for the data shown in panels A–D. Circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test; for details about boxplots see Materials and Methods ). One or two asterisks denote statistically significant differences between the medians of two distributions (

    Article Snippet: HMEC-1 cells were treated with control or experimental siRNA as described above. mRNA was harvested using the RNeasy Micro Kit (Qiagen; 74004), and cDNA was prepared using the Superscript III First-strand Synthesis SuperMix (ThermoFisher; 18080–400).

    Techniques: Infection, Flow Cytometry, Cytometry, Fluorescence, MANN-WHITNEY

    Lm adhesion, but not invasion efficiency, is increased when HMEC-1 reside on stiff hydrogels. HMEC-1 residing on soft (3-kPa) or stiff (70-kPa) PA hydrogels and treated with vehicle control or 2 μM PF537228 FAK inhibitor were infected with Lm (constitutively expressing GFP) at an MOI between 1.5 and 15. At 30 min postinfection, samples were fixed and immunostained, and infection was analyzed by microscopy followed by image processing. Boxplots show (A) total bacteria per cell; (B) internalized bacteria per cell; (C) invasion efficiency (ratio of internalized bacteria to total bacteria); (D) cells in the field of view. Representative data come from one of three independent experiments. N = 800–1000 cells were analyzed for each condition. Two asterisks denote statistically significant differences between the medians of two distributions (

    Journal: Molecular Biology of the Cell

    Article Title: Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin

    doi: 10.1091/mbc.E18-04-0228

    Figure Lengend Snippet: Lm adhesion, but not invasion efficiency, is increased when HMEC-1 reside on stiff hydrogels. HMEC-1 residing on soft (3-kPa) or stiff (70-kPa) PA hydrogels and treated with vehicle control or 2 μM PF537228 FAK inhibitor were infected with Lm (constitutively expressing GFP) at an MOI between 1.5 and 15. At 30 min postinfection, samples were fixed and immunostained, and infection was analyzed by microscopy followed by image processing. Boxplots show (A) total bacteria per cell; (B) internalized bacteria per cell; (C) invasion efficiency (ratio of internalized bacteria to total bacteria); (D) cells in the field of view. Representative data come from one of three independent experiments. N = 800–1000 cells were analyzed for each condition. Two asterisks denote statistically significant differences between the medians of two distributions (

    Article Snippet: HMEC-1 cells were treated with control or experimental siRNA as described above. mRNA was harvested using the RNeasy Micro Kit (Qiagen; 74004), and cDNA was prepared using the Superscript III First-strand Synthesis SuperMix (ThermoFisher; 18080–400).

    Techniques: Infection, Expressing, Microscopy

    Lower FAK activity leads to reduced amount of cell surface vimentin. (A, B) 2D-PAGE gels of plasma membrane proteins of HMEC-1 grown on TC polystyrene substrates treated for 1 h with vehicle control (A) or 2 µM PF537228 FAK inhibitor (B). pH increases from left to right. Gels were silver-stained and one isoelectric point marker (tropomyosin), added to each sample as an internal standard, is marked with a black arrow. The one spot that differed consistently between three independent experiments is indicated with a black circle and corresponds to vimentin (55 kDa).

    Journal: Molecular Biology of the Cell

    Article Title: Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin

    doi: 10.1091/mbc.E18-04-0228

    Figure Lengend Snippet: Lower FAK activity leads to reduced amount of cell surface vimentin. (A, B) 2D-PAGE gels of plasma membrane proteins of HMEC-1 grown on TC polystyrene substrates treated for 1 h with vehicle control (A) or 2 µM PF537228 FAK inhibitor (B). pH increases from left to right. Gels were silver-stained and one isoelectric point marker (tropomyosin), added to each sample as an internal standard, is marked with a black arrow. The one spot that differed consistently between three independent experiments is indicated with a black circle and corresponds to vimentin (55 kDa).

    Article Snippet: HMEC-1 cells were treated with control or experimental siRNA as described above. mRNA was harvested using the RNeasy Micro Kit (Qiagen; 74004), and cDNA was prepared using the Superscript III First-strand Synthesis SuperMix (ThermoFisher; 18080–400).

    Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis, Staining, Marker

    Blocking HMEC-1 with anti-vimentin antibody reduces Li adhesion onto HMEC-1 but not uptake of beads. (A) Boxplots showing the number of bacteria per cell, for HMEC-1 residing on glass substrates and treated with vehicle control, 2 μM PF537228 FAK inhibitor, or 80 μg/ml H-84 anti-vimentin antibody prior to infection. Cells were infected with Lm or Li at an MOI of 4. At 30 min postinfection, samples were fixed and immunostained and adhesion of bacteria was analyzed by microscopy followed by image processing. For each condition, 2300–2600 cells were analyzed in total and data refer to one of two independent experiments. Two asterisks denote statistically significant differences between the median values of control cells vs. all other groups (

    Journal: Molecular Biology of the Cell

    Article Title: Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin

    doi: 10.1091/mbc.E18-04-0228

    Figure Lengend Snippet: Blocking HMEC-1 with anti-vimentin antibody reduces Li adhesion onto HMEC-1 but not uptake of beads. (A) Boxplots showing the number of bacteria per cell, for HMEC-1 residing on glass substrates and treated with vehicle control, 2 μM PF537228 FAK inhibitor, or 80 μg/ml H-84 anti-vimentin antibody prior to infection. Cells were infected with Lm or Li at an MOI of 4. At 30 min postinfection, samples were fixed and immunostained and adhesion of bacteria was analyzed by microscopy followed by image processing. For each condition, 2300–2600 cells were analyzed in total and data refer to one of two independent experiments. Two asterisks denote statistically significant differences between the median values of control cells vs. all other groups (

    Article Snippet: HMEC-1 cells were treated with control or experimental siRNA as described above. mRNA was harvested using the RNeasy Micro Kit (Qiagen; 74004), and cDNA was prepared using the Superscript III First-strand Synthesis SuperMix (ThermoFisher; 18080–400).

    Techniques: Blocking Assay, Infection, Microscopy

    FAK activity of HMEC-1 residing on soft PA hydrogels is decreased, as is Lm uptake. (A) Western blots from whole HMEC-1 lysates showing expression of phosphorylated FAK (Tyr397) and total FAK for cells residing on soft gels (3 kPa), stiff gels (70 kPa), and TC polystyrene substrates with or without 2 μM PF537228 FAK inhibitor. In each Western blot, equal quantities of protein were loaded and equal loading was confirmed in relation to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. In each case, the Western blots shown are representative of three independent experiments. (B, C) Normalized ratios of FAK/GAPDH (B) and pFAK (Tyr397)/GAPDH (C) for HMEC-1 residing on varying-stiffness substrates and treated or not with 2 μM PF537228 FAK inhibitor. Different color circles correspond to data from three independent experiments. Black bars represent the means of the three independent experiments. For each experiment, values have been normalized relative to the ratio for cells residing on polystyrene substrates. (D) Inhibition of bacterial uptake by FAK inhibitors. FAK-14, PF573228, or vehicle control was added 1 h before addition of bacteria to HMEC-1 residing on polystyrene substrates. Percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of inhibitor concentration (mean ± SD, N = 4 replicates). x = 0 corresponds to cells treated with vehicle control. Inset shows the same data with concentration on a log scale. Infection was analyzed by flow cytometry, 7–8 h after infection. MOI is 80. Representative data come from one of three independent experiments. (E) Boxplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or FAK siRNA (siFAK) (means ± SD, three independent experiments and N = 6 replicates per experiment). MOI is 60 (gray) or 20 (green). Circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test; for details about boxplots see Materials and Methods ). One or two asterisks denote statistically significant differences between the medians of two distributions (

    Journal: Molecular Biology of the Cell

    Article Title: Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin

    doi: 10.1091/mbc.E18-04-0228

    Figure Lengend Snippet: FAK activity of HMEC-1 residing on soft PA hydrogels is decreased, as is Lm uptake. (A) Western blots from whole HMEC-1 lysates showing expression of phosphorylated FAK (Tyr397) and total FAK for cells residing on soft gels (3 kPa), stiff gels (70 kPa), and TC polystyrene substrates with or without 2 μM PF537228 FAK inhibitor. In each Western blot, equal quantities of protein were loaded and equal loading was confirmed in relation to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. In each case, the Western blots shown are representative of three independent experiments. (B, C) Normalized ratios of FAK/GAPDH (B) and pFAK (Tyr397)/GAPDH (C) for HMEC-1 residing on varying-stiffness substrates and treated or not with 2 μM PF537228 FAK inhibitor. Different color circles correspond to data from three independent experiments. Black bars represent the means of the three independent experiments. For each experiment, values have been normalized relative to the ratio for cells residing on polystyrene substrates. (D) Inhibition of bacterial uptake by FAK inhibitors. FAK-14, PF573228, or vehicle control was added 1 h before addition of bacteria to HMEC-1 residing on polystyrene substrates. Percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of inhibitor concentration (mean ± SD, N = 4 replicates). x = 0 corresponds to cells treated with vehicle control. Inset shows the same data with concentration on a log scale. Infection was analyzed by flow cytometry, 7–8 h after infection. MOI is 80. Representative data come from one of three independent experiments. (E) Boxplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or FAK siRNA (siFAK) (means ± SD, three independent experiments and N = 6 replicates per experiment). MOI is 60 (gray) or 20 (green). Circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test; for details about boxplots see Materials and Methods ). One or two asterisks denote statistically significant differences between the medians of two distributions (

    Article Snippet: HMEC-1 cells were treated with control or experimental siRNA as described above. mRNA was harvested using the RNeasy Micro Kit (Qiagen; 74004), and cDNA was prepared using the Superscript III First-strand Synthesis SuperMix (ThermoFisher; 18080–400).

    Techniques: Activity Assay, Western Blot, Expressing, Inhibition, Infection, Concentration Assay, Flow Cytometry, Cytometry, MANN-WHITNEY

    Surface vimentin of HMEC-1 is implicated in Lm uptake. (A) Decrease in bacterial uptake after blocking HMEC-1 with anti-vimentin antibody H-84. Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of antibody concentration (means ± SD and N = 6 replicates per experiment). Representative data come from one of three independent experiments. Infection was analyzed by flow cytometry, 7–8 h after infection. (B) Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or with vimentin siRNA (siVIM) (means ± SD, and N = 6 replicates per experiment). Representative data come from one of three independent experiments. MOI is 50 (black barplots) and 17 (gray barplots). (C) Decreased uptake of Lm when HMEC-1 are treated with withaferin that captures soluble vimentin 30 min prior to infection. Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of withaferin concentration (means ± SD and N = 6 replicates per experiment). Representative data come from one of three independent experiments. Infection was analyzed by flow cytometry 7–8 h after infection. (D) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: Δ actA (black), Δ actA/ Δ inlB (gray; actAp::mTagRFP), and HMEC-1 were treated with vehicle control (circle) or withaferin (diamond) for 30 min prior to infection. The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. MOI ranged from 50 to120. Two asterisks denote statistically significant differences between the medians of infection fraction of control vs. all other groups ( p

    Journal: Molecular Biology of the Cell

    Article Title: Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin

    doi: 10.1091/mbc.E18-04-0228

    Figure Lengend Snippet: Surface vimentin of HMEC-1 is implicated in Lm uptake. (A) Decrease in bacterial uptake after blocking HMEC-1 with anti-vimentin antibody H-84. Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of antibody concentration (means ± SD and N = 6 replicates per experiment). Representative data come from one of three independent experiments. Infection was analyzed by flow cytometry, 7–8 h after infection. (B) Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or with vimentin siRNA (siVIM) (means ± SD, and N = 6 replicates per experiment). Representative data come from one of three independent experiments. MOI is 50 (black barplots) and 17 (gray barplots). (C) Decreased uptake of Lm when HMEC-1 are treated with withaferin that captures soluble vimentin 30 min prior to infection. Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of withaferin concentration (means ± SD and N = 6 replicates per experiment). Representative data come from one of three independent experiments. Infection was analyzed by flow cytometry 7–8 h after infection. (D) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: Δ actA (black), Δ actA/ Δ inlB (gray; actAp::mTagRFP), and HMEC-1 were treated with vehicle control (circle) or withaferin (diamond) for 30 min prior to infection. The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. MOI ranged from 50 to120. Two asterisks denote statistically significant differences between the medians of infection fraction of control vs. all other groups ( p

    Article Snippet: HMEC-1 cells were treated with control or experimental siRNA as described above. mRNA was harvested using the RNeasy Micro Kit (Qiagen; 74004), and cDNA was prepared using the Superscript III First-strand Synthesis SuperMix (ThermoFisher; 18080–400).

    Techniques: Blocking Assay, Infection, Concentration Assay, Flow Cytometry, Cytometry

    curcumin (Cur) impairs endothelin-1 (ET-1) production in Trypanosoma cruzi -infected human microvascular endothelial cells (HMEC-1) through downregulation of Ca 2+ -sensitive NFAT signalling. The involvement of Ca 2+ -dependent mechanisms in T. cruzi -promoted ET-1 release from infected HMEC-1 was verified. (A) Soluble ET-1 levels in the media of uninfected (PBS) or infected ( T. cruzi ) cells, cultured in the presence or in the absence of Cur at different concentrations (13.5 and 27.0 μM), were measured by enzyme-linked immunosorbent assay (ELISA). In some experiments, the cells were pre-treated for 1 h with chemical inhibitors of NFAT or c-Src kinase-mediated pathways (5 μM CsA or 20 μM Src-I1, respectively), and their effect on ET-1 secretion was further determined. Data are the means ± standard deviations of three independent experiments, each performed in triplicate. *p

    Journal: Memórias do Instituto Oswaldo Cruz

    Article Title: Curcumin exerts anti-inflammatory and vasoprotective effects through amelioration of NFAT-dependent endothelin-1 production in mice with acute Chagas cardiomyopathy

    doi: 10.1590/0074-02760180171

    Figure Lengend Snippet: curcumin (Cur) impairs endothelin-1 (ET-1) production in Trypanosoma cruzi -infected human microvascular endothelial cells (HMEC-1) through downregulation of Ca 2+ -sensitive NFAT signalling. The involvement of Ca 2+ -dependent mechanisms in T. cruzi -promoted ET-1 release from infected HMEC-1 was verified. (A) Soluble ET-1 levels in the media of uninfected (PBS) or infected ( T. cruzi ) cells, cultured in the presence or in the absence of Cur at different concentrations (13.5 and 27.0 μM), were measured by enzyme-linked immunosorbent assay (ELISA). In some experiments, the cells were pre-treated for 1 h with chemical inhibitors of NFAT or c-Src kinase-mediated pathways (5 μM CsA or 20 μM Src-I1, respectively), and their effect on ET-1 secretion was further determined. Data are the means ± standard deviations of three independent experiments, each performed in triplicate. *p

    Article Snippet: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) - Total RNA was extracted from HMEC-1/myocardial cells and treated with DNase I using the RNeasy® Micro kit (Qiagen, Hilden, Germany). mRNA levels of human and murine ET-1 were determined by qRT-PCR using cDNA, obtained from the reverse transcription reactions, as template, with MyiQ™ Single-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA) and HotStart-IT® SYBR® Green One-Step qRT-PCR Master Mix Kit (Affymetrix, Santa Clara, CA, USA).

    Techniques: Infection, Cell Culture, Enzyme-linked Immunosorbent Assay

    Cluster analysis of the gene expression. Clustering of differentially expressed genes in the HMEC-1 cells infected with LV-Con or LV-shGPR4.

    Journal: International Journal of Clinical and Experimental Medicine

    Article Title: RNAi targeting GPR4 influences HMEC-1 gene expression by microarray analysis

    doi:

    Figure Lengend Snippet: Cluster analysis of the gene expression. Clustering of differentially expressed genes in the HMEC-1 cells infected with LV-Con or LV-shGPR4.

    Article Snippet: LV-eGFP and LV-shGPR4 infected HMEC-1 cells were harvested and total RNA was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions.

    Techniques: Expressing, Infection