ecis model zθ  (Applied BioPhysics)


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    Structured Review

    Applied BioPhysics ecis model zθ
    Role of Junctions in Opto-RhoGEF induced changes in vascular barrier strength. ( A )Linearity Index for a BOEC monolayer stably expressing Lck-mTurquoise2-iLID and either SspB-HaloTag-TIAM1(DHPH) (purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) for the frame before photo activation (pre) and the last frame of photo activation (Activation). Images of the junctions are in Fig. S2.2 and example of linearity index analysis in Fig. S3.1A . Each dot represents and individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The number of cells is: Opto-TIAM pre=55, Opto-TIAM Activation=52, Opto-ITSN pre=71, Opto-ITSN Activation=76, Opto-P63 pre=74, Opto-P63 Activation=51. The data is from two independent experiments. ( B ) Resistance of a monolayer of BOECs stably expressing Lck-mTurquoise2-iLID, solely as a control (grey), and either SspB-HaloTag-TIAM1(DHPH)(purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) measured with <t>ECIS</t> at 4000 Hz every 10 s. Cyan bars indicated photo activation with blue LED light (60 min, 15 min). Gray bar with dashed lines indicates the addition of VE-cadherin blocking antibody in medium (darker color line) or medium as a control (lighter color line). At the end of the gray bar the medium is replaced for all conditions. Thin lines represent the average value from one well of an 8W10E PET ECIS array. Thick lines represent the mean. Four wells were measured for each condition. ( C ) Representative zoom ins from confocal microscopy images of a BOEC monolayer stably expressing Lck-mTurquoise2-iLID (not shown) and either SspB-HaloTag-TIAM1(DHPH)/ ITSN1(DHPH) or p63RhoGEF(DH) stained with JF552 nm dye (LUT = mpl-magma, bright colors indicating higher intensity). Additionally, stained for VE-Cadherin with the live labeling antibody Alexa Fluor 647 Mouse Anti-Human CD144 (white in merge, grey inverted in single channel). Left panel shows untreated cells, right panel shows cells treated with the VE-cadherin blocking antibody. Scale bars: 25 µm. Times are min:s from the start of the recording. Gray bar indicates the condition before photo activation. Cyan bar indicates 442 nm photo activation. Arrows indicate overlap and protrusions. Asterisks indicate holes in monolayer.
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    Images

    1) Product Images from "Opto-RhoGEFs: an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength"

    Article Title: Opto-RhoGEFs: an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength

    Journal: bioRxiv

    doi: 10.1101/2022.10.17.512253

    Role of Junctions in Opto-RhoGEF induced changes in vascular barrier strength. ( A )Linearity Index for a BOEC monolayer stably expressing Lck-mTurquoise2-iLID and either SspB-HaloTag-TIAM1(DHPH) (purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) for the frame before photo activation (pre) and the last frame of photo activation (Activation). Images of the junctions are in Fig. S2.2 and example of linearity index analysis in Fig. S3.1A . Each dot represents and individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The number of cells is: Opto-TIAM pre=55, Opto-TIAM Activation=52, Opto-ITSN pre=71, Opto-ITSN Activation=76, Opto-P63 pre=74, Opto-P63 Activation=51. The data is from two independent experiments. ( B ) Resistance of a monolayer of BOECs stably expressing Lck-mTurquoise2-iLID, solely as a control (grey), and either SspB-HaloTag-TIAM1(DHPH)(purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) measured with ECIS at 4000 Hz every 10 s. Cyan bars indicated photo activation with blue LED light (60 min, 15 min). Gray bar with dashed lines indicates the addition of VE-cadherin blocking antibody in medium (darker color line) or medium as a control (lighter color line). At the end of the gray bar the medium is replaced for all conditions. Thin lines represent the average value from one well of an 8W10E PET ECIS array. Thick lines represent the mean. Four wells were measured for each condition. ( C ) Representative zoom ins from confocal microscopy images of a BOEC monolayer stably expressing Lck-mTurquoise2-iLID (not shown) and either SspB-HaloTag-TIAM1(DHPH)/ ITSN1(DHPH) or p63RhoGEF(DH) stained with JF552 nm dye (LUT = mpl-magma, bright colors indicating higher intensity). Additionally, stained for VE-Cadherin with the live labeling antibody Alexa Fluor 647 Mouse Anti-Human CD144 (white in merge, grey inverted in single channel). Left panel shows untreated cells, right panel shows cells treated with the VE-cadherin blocking antibody. Scale bars: 25 µm. Times are min:s from the start of the recording. Gray bar indicates the condition before photo activation. Cyan bar indicates 442 nm photo activation. Arrows indicate overlap and protrusions. Asterisks indicate holes in monolayer.
    Figure Legend Snippet: Role of Junctions in Opto-RhoGEF induced changes in vascular barrier strength. ( A )Linearity Index for a BOEC monolayer stably expressing Lck-mTurquoise2-iLID and either SspB-HaloTag-TIAM1(DHPH) (purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) for the frame before photo activation (pre) and the last frame of photo activation (Activation). Images of the junctions are in Fig. S2.2 and example of linearity index analysis in Fig. S3.1A . Each dot represents and individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The number of cells is: Opto-TIAM pre=55, Opto-TIAM Activation=52, Opto-ITSN pre=71, Opto-ITSN Activation=76, Opto-P63 pre=74, Opto-P63 Activation=51. The data is from two independent experiments. ( B ) Resistance of a monolayer of BOECs stably expressing Lck-mTurquoise2-iLID, solely as a control (grey), and either SspB-HaloTag-TIAM1(DHPH)(purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) measured with ECIS at 4000 Hz every 10 s. Cyan bars indicated photo activation with blue LED light (60 min, 15 min). Gray bar with dashed lines indicates the addition of VE-cadherin blocking antibody in medium (darker color line) or medium as a control (lighter color line). At the end of the gray bar the medium is replaced for all conditions. Thin lines represent the average value from one well of an 8W10E PET ECIS array. Thick lines represent the mean. Four wells were measured for each condition. ( C ) Representative zoom ins from confocal microscopy images of a BOEC monolayer stably expressing Lck-mTurquoise2-iLID (not shown) and either SspB-HaloTag-TIAM1(DHPH)/ ITSN1(DHPH) or p63RhoGEF(DH) stained with JF552 nm dye (LUT = mpl-magma, bright colors indicating higher intensity). Additionally, stained for VE-Cadherin with the live labeling antibody Alexa Fluor 647 Mouse Anti-Human CD144 (white in merge, grey inverted in single channel). Left panel shows untreated cells, right panel shows cells treated with the VE-cadherin blocking antibody. Scale bars: 25 µm. Times are min:s from the start of the recording. Gray bar indicates the condition before photo activation. Cyan bar indicates 442 nm photo activation. Arrows indicate overlap and protrusions. Asterisks indicate holes in monolayer.

    Techniques Used: Stable Transfection, Expressing, Activation Assay, Electric Cell-substrate Impedance Sensing, Blocking Assay, Positron Emission Tomography, Confocal Microscopy, Staining, Labeling

    Photoactivation of Opto-RhoGEFs controls permeability and vascular barrier strength. ( A ) Fluorescence intensity measured in a transwell assay for a monolayer of BOECs stably expressing Lck-mTurquoise2-iLID, solely as a control, and SspB-HaloTag-TIAM1(DHPH) treated with FITC 0.3 kDa, FITC dextran 10 kDa and FITC dextran 70 kDa. Photo activated with blue LED light for 10 min as indicated by cyan background in the graph or kept in the dark indicated by white background. Dots represent individual transwell dishes. The black bar indicates the mean. The number of transwell dishes per condition is: Control_Lck-iLID=9, OpotTIAM_dark=6, OptoTIAM_light=9. The data is from three experiments. ( B ) Resistance of a monolayer of BOECs stably expressing Lck-mTurquoise2-iLID, solely as a control (grey), and either SspB-HaloTag-TIAM1(DHPH)(purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) measured with ECIS at 4000 Hz every 10 s. Cyan bars indicated photo activation with blue LED light (1 min, 5 min, 10 min, 3x 15 min, 120 min). Thin lines represent the average value from one well of an 8W10E PET ECIS array. Thick lines represent the mean. The number of wells per condition was: OptoTIAM=6, OptoITSN=6, OptoP63=6, control=4. ( C ) Representative zoom ins from confocal microscopy images of a BOEC monolayer stably expressing Lck-mTurquoise2-iLID (not shown) and either SspB-HaloTag-TIAM1(DHPH)/ ITSN1(DHPH) or p63RhoGEF(DH) stained with JF552 nm dye (LUT = mpl-magma, bright colors indicating higher intensity). Additionally, stained for VE-Cadherin with the live labeling antibody Alexa Fluor 647 Mouse Anti-Human CD144 (white). Scale bars: 25 µm. Times are min:s from the start of the recording. Cyan bar indicates 442 nm photo activation. Arrows indicate overlap and protrusions. Asterisks indicate holes in monolayer. Whole field of view is shown in S4.2 .
    Figure Legend Snippet: Photoactivation of Opto-RhoGEFs controls permeability and vascular barrier strength. ( A ) Fluorescence intensity measured in a transwell assay for a monolayer of BOECs stably expressing Lck-mTurquoise2-iLID, solely as a control, and SspB-HaloTag-TIAM1(DHPH) treated with FITC 0.3 kDa, FITC dextran 10 kDa and FITC dextran 70 kDa. Photo activated with blue LED light for 10 min as indicated by cyan background in the graph or kept in the dark indicated by white background. Dots represent individual transwell dishes. The black bar indicates the mean. The number of transwell dishes per condition is: Control_Lck-iLID=9, OpotTIAM_dark=6, OptoTIAM_light=9. The data is from three experiments. ( B ) Resistance of a monolayer of BOECs stably expressing Lck-mTurquoise2-iLID, solely as a control (grey), and either SspB-HaloTag-TIAM1(DHPH)(purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) measured with ECIS at 4000 Hz every 10 s. Cyan bars indicated photo activation with blue LED light (1 min, 5 min, 10 min, 3x 15 min, 120 min). Thin lines represent the average value from one well of an 8W10E PET ECIS array. Thick lines represent the mean. The number of wells per condition was: OptoTIAM=6, OptoITSN=6, OptoP63=6, control=4. ( C ) Representative zoom ins from confocal microscopy images of a BOEC monolayer stably expressing Lck-mTurquoise2-iLID (not shown) and either SspB-HaloTag-TIAM1(DHPH)/ ITSN1(DHPH) or p63RhoGEF(DH) stained with JF552 nm dye (LUT = mpl-magma, bright colors indicating higher intensity). Additionally, stained for VE-Cadherin with the live labeling antibody Alexa Fluor 647 Mouse Anti-Human CD144 (white). Scale bars: 25 µm. Times are min:s from the start of the recording. Cyan bar indicates 442 nm photo activation. Arrows indicate overlap and protrusions. Asterisks indicate holes in monolayer. Whole field of view is shown in S4.2 .

    Techniques Used: Permeability, Fluorescence, Transwell Assay, Stable Transfection, Expressing, Electric Cell-substrate Impedance Sensing, Activation Assay, Positron Emission Tomography, Confocal Microscopy, Staining, Labeling

    2) Product Images from "Aging-regulated TUG1 is dispensable for endothelial cell function"

    Article Title: Aging-regulated TUG1 is dispensable for endothelial cell function

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0265160

    TUG1 is not important for basal cell turnover, barrier or mitochondrial function, migration and monocyte adhesion. (A)–(G) HUVECs were transfected with two LNA GapmeRs against TUG1 —LNA TUG1_1 and LNA TUG1_2 –and LNA Ctrl (10 nM) and (A) expression levels were measured after 48 hours by RT-qPCR. Expression is relative to GAPDH (n = 4; SEM; RM one-way ANOVA with Greenhouse-Geisser correction and Sidak multiple comparison test). (B) Relative cell growth determined from cell count at 0 h, 24 h, 48 h and 72 h (n = 3; SEM; RM Two-way ANOVA with Tuckey multiple comparison test). (C) Caspase-3/7 activity was measured by determination of fluorescence with ELISA plate reader (n = 3; SEM; One-way ANOVA with Holm-Sidak correction). Staurosporine was taken along as a postive control. (D) Cell-cell interactions (Rb) and cell-matrix-interactions (α) were measured by Electric Cell Impedance Sensing (ECIS; n = 3; SEM; Kruskal-Wallis-test with Dunn´s correction). (E) Determination of re-establishment of monolayer after wounding using ECIS (n = 3; SEM; One-way ANOVA with Holm-Sidak multiple comparison test). (F) Seahorse mitochondrial stress test assessing multiple mitochondrial characteristics via measurement of changes in Oxygen Consumption Rate (OCR) after serial injection of Oligomycin, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) and Rotenone A/Antimycin (n = 3; SEM; One-way ANOVA with Holm-Sidak multiple comparison test. One representative experiment displaying the changes of OCR throughout the progress of the Seahorse mitochondrial stress test assay. (G) Assessment of monocyte adhesion with and without TNF-α stimulation. (n = 3; SEM; Two-way ANOVA with Tuckey multiple comparison test).
    Figure Legend Snippet: TUG1 is not important for basal cell turnover, barrier or mitochondrial function, migration and monocyte adhesion. (A)–(G) HUVECs were transfected with two LNA GapmeRs against TUG1 —LNA TUG1_1 and LNA TUG1_2 –and LNA Ctrl (10 nM) and (A) expression levels were measured after 48 hours by RT-qPCR. Expression is relative to GAPDH (n = 4; SEM; RM one-way ANOVA with Greenhouse-Geisser correction and Sidak multiple comparison test). (B) Relative cell growth determined from cell count at 0 h, 24 h, 48 h and 72 h (n = 3; SEM; RM Two-way ANOVA with Tuckey multiple comparison test). (C) Caspase-3/7 activity was measured by determination of fluorescence with ELISA plate reader (n = 3; SEM; One-way ANOVA with Holm-Sidak correction). Staurosporine was taken along as a postive control. (D) Cell-cell interactions (Rb) and cell-matrix-interactions (α) were measured by Electric Cell Impedance Sensing (ECIS; n = 3; SEM; Kruskal-Wallis-test with Dunn´s correction). (E) Determination of re-establishment of monolayer after wounding using ECIS (n = 3; SEM; One-way ANOVA with Holm-Sidak multiple comparison test). (F) Seahorse mitochondrial stress test assessing multiple mitochondrial characteristics via measurement of changes in Oxygen Consumption Rate (OCR) after serial injection of Oligomycin, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) and Rotenone A/Antimycin (n = 3; SEM; One-way ANOVA with Holm-Sidak multiple comparison test. One representative experiment displaying the changes of OCR throughout the progress of the Seahorse mitochondrial stress test assay. (G) Assessment of monocyte adhesion with and without TNF-α stimulation. (n = 3; SEM; Two-way ANOVA with Tuckey multiple comparison test).

    Techniques Used: Migration, Transfection, Expressing, Quantitative RT-PCR, Cell Counting, Activity Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Electric Cell-substrate Impedance Sensing, Injection

    3) Product Images from "NF-κB-dependent repression of Sox18 transcription factor requires the epigenetic regulators histone deacetylases 1 and 2 in acute lung injury"

    Article Title: NF-κB-dependent repression of Sox18 transcription factor requires the epigenetic regulators histone deacetylases 1 and 2 in acute lung injury

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2022.947537

    Effects of class I HDACs selective inhibitors on LPS-induced endothelial barrier disruption in cultured human lung microvascular endothelial cells. HLMVEC monolayers grown in ECIS arrays were pre-treated with HDAC inhibitors or vehicle for 30 min then challenged with LPS. TER was recorded in real time to evaluate possible barrier-protective effects of HDAC inhibitors. Data are shown for tacedinaline (HDAC -1 and -3 inhibitor) (A) , FK228 (HDAC -1 and -2 inhibitor) (B) , MI192 (HDAC -2 and -3 inhibitor) (C) , and RGFP966 (HDAC3 inhibitor) (D) . Data are mean ± SEM, n = 4. * p
    Figure Legend Snippet: Effects of class I HDACs selective inhibitors on LPS-induced endothelial barrier disruption in cultured human lung microvascular endothelial cells. HLMVEC monolayers grown in ECIS arrays were pre-treated with HDAC inhibitors or vehicle for 30 min then challenged with LPS. TER was recorded in real time to evaluate possible barrier-protective effects of HDAC inhibitors. Data are shown for tacedinaline (HDAC -1 and -3 inhibitor) (A) , FK228 (HDAC -1 and -2 inhibitor) (B) , MI192 (HDAC -2 and -3 inhibitor) (C) , and RGFP966 (HDAC3 inhibitor) (D) . Data are mean ± SEM, n = 4. * p

    Techniques Used: Cell Culture, Electric Cell-substrate Impedance Sensing

    4) Product Images from "Endothelial Barrier Disruption by Lipid Emulsions Containing a High Amount of N3 Fatty Acids (Omegaven) but Not N6 Fatty Acids (Intralipid)"

    Article Title: Endothelial Barrier Disruption by Lipid Emulsions Containing a High Amount of N3 Fatty Acids (Omegaven) but Not N6 Fatty Acids (Intralipid)

    Journal: Cells

    doi: 10.3390/cells11142202

    Omegaven ® reduces the barrier formation at high concentrations. Omegaven ® and Intralipid ® lipid emulsions at 0.01, 0.1 and 1 mM concentrations were added to endothelial cells during cell seeding on the ECIS electrode ( A ), during the logarithmic growth phase ( B ), and in cells of the plateau growth phase ( C ). The barrier formation was monitored by an ECIS ® Zθ (Z Theta) device, and 60 measurements of resistance (ohm) in 70 h were recorded for cells grown in triplicates. In addition, areas under the curve ( D ) for the graph presented in ( A – C ) were analyzed using (GraphPad, San Diego, CA, USA), X.Y. analysis/Area under curve function.
    Figure Legend Snippet: Omegaven ® reduces the barrier formation at high concentrations. Omegaven ® and Intralipid ® lipid emulsions at 0.01, 0.1 and 1 mM concentrations were added to endothelial cells during cell seeding on the ECIS electrode ( A ), during the logarithmic growth phase ( B ), and in cells of the plateau growth phase ( C ). The barrier formation was monitored by an ECIS ® Zθ (Z Theta) device, and 60 measurements of resistance (ohm) in 70 h were recorded for cells grown in triplicates. In addition, areas under the curve ( D ) for the graph presented in ( A – C ) were analyzed using (GraphPad, San Diego, CA, USA), X.Y. analysis/Area under curve function.

    Techniques Used: Electric Cell-substrate Impedance Sensing

    Omegaven ® increases the capacitance indicating reduced electrode coverage. Omegaven ® and Intralipid ® lipid emulsions at 0.01, 0.1 and 1 mM concentrations were added to endothelial cells during cell seeding on the ECIS electrode ( A ), in the cells logarithmic growth phase ( B ), and in cells plateau growth phase ( C ). The electrode coverage was monitored by an ECIS ® Zθ (Z Theta) device, and 60 measurements of capacitance (nF) in 70 h were recorded for cells grown in triplicates. The decrease in capacitance of cells grown on the electrode is directly proportional to the electrode coverage. Therefore, the higher values indicate the lower electrode coverage by cells. For the graph presented in ( A – C ), areas under the curve ( D ) were analyzed using (GraphPad, San Diego, CA, USA), X.Y. analysis/Area under curve function.
    Figure Legend Snippet: Omegaven ® increases the capacitance indicating reduced electrode coverage. Omegaven ® and Intralipid ® lipid emulsions at 0.01, 0.1 and 1 mM concentrations were added to endothelial cells during cell seeding on the ECIS electrode ( A ), in the cells logarithmic growth phase ( B ), and in cells plateau growth phase ( C ). The electrode coverage was monitored by an ECIS ® Zθ (Z Theta) device, and 60 measurements of capacitance (nF) in 70 h were recorded for cells grown in triplicates. The decrease in capacitance of cells grown on the electrode is directly proportional to the electrode coverage. Therefore, the higher values indicate the lower electrode coverage by cells. For the graph presented in ( A – C ), areas under the curve ( D ) were analyzed using (GraphPad, San Diego, CA, USA), X.Y. analysis/Area under curve function.

    Techniques Used: Electric Cell-substrate Impedance Sensing

    5) Product Images from "The splicing‐regulatory lncRNA NTRAS sustains vascular integrity"

    Article Title: The splicing‐regulatory lncRNA NTRAS sustains vascular integrity

    Journal: EMBO Reports

    doi: 10.15252/embr.202154157

    NTRAS controls TJP1 splicing and endothelial barrier function Scheme depicting the in vitro splicing of a TJP1 exon 19–exon 20 splice substrate featuring an intronic splicing silencer (ISS). hnRNPL binding sites are highlighted in red. In vitro splicing efficiency of the TJP1 splice substrate, comparing mock, NTRAS‐depleted, and NTRAS‐CA 16 motif add‐back conditions ( n = 7–12 independent biological replicates). Co‐precipitation of TJP1 pre‐mRNA in anti‐hnRNPL RIPs, using nuclear lysates from control and NTRAS‐silenced HUVECs ( n = 6 independent biological replicates). Co‐precipitation of TJP1 pre‐mRNA in anti‐hnRNPL RIPs, using nuclear lysates from control and NTRAS‐overexpressing cells ( n = 5 independent biological replicates). Representative western blot on the right. Co‐precipitation of TJP1 pre‐mRNA in anti‐hnRNPL RIPs, using nuclear lysates from control and NTRAS‐CA 16 motif overexpressing cells ( n = 5 independent biological replicates). Representative western blot on the right. RT–PCR‐based analysis of TJP1 exon 20 inclusion upon NTRAS overexpression and overexpression of the NTRAS‐CA 16 motif ( n = 8 independent biological replicates). Endothelial resistance of NTRAS‐silenced HUVECs ( n = 3–5 independent biological replicates), analyzed by electrical cell‐substrate impedance sensing (ECIS). Endothelial resistance of hnRNPL‐silenced HUVECs ( n = 3 independent biological replicates), analyzed by ECIS. Analysis of TJP1 exon 20 inclusion by RT–PCR upon transfection of HUVECs with a control SSO or an SSO masking the exon 20–intron 20 boundary (E20 SSO) ( n = 9 independent biological replicates). Representative agarose gel on the right. Schematic outline at the top right. Endothelial resistance of control SSO‐ or E20 SSO‐transfected HUVECs ( n = 4 independent biological replicates), analyzed by ECIS. Data information: In (B–J), data are represented as mean ± SEM. n.s.: non‐significant, * P
    Figure Legend Snippet: NTRAS controls TJP1 splicing and endothelial barrier function Scheme depicting the in vitro splicing of a TJP1 exon 19–exon 20 splice substrate featuring an intronic splicing silencer (ISS). hnRNPL binding sites are highlighted in red. In vitro splicing efficiency of the TJP1 splice substrate, comparing mock, NTRAS‐depleted, and NTRAS‐CA 16 motif add‐back conditions ( n = 7–12 independent biological replicates). Co‐precipitation of TJP1 pre‐mRNA in anti‐hnRNPL RIPs, using nuclear lysates from control and NTRAS‐silenced HUVECs ( n = 6 independent biological replicates). Co‐precipitation of TJP1 pre‐mRNA in anti‐hnRNPL RIPs, using nuclear lysates from control and NTRAS‐overexpressing cells ( n = 5 independent biological replicates). Representative western blot on the right. Co‐precipitation of TJP1 pre‐mRNA in anti‐hnRNPL RIPs, using nuclear lysates from control and NTRAS‐CA 16 motif overexpressing cells ( n = 5 independent biological replicates). Representative western blot on the right. RT–PCR‐based analysis of TJP1 exon 20 inclusion upon NTRAS overexpression and overexpression of the NTRAS‐CA 16 motif ( n = 8 independent biological replicates). Endothelial resistance of NTRAS‐silenced HUVECs ( n = 3–5 independent biological replicates), analyzed by electrical cell‐substrate impedance sensing (ECIS). Endothelial resistance of hnRNPL‐silenced HUVECs ( n = 3 independent biological replicates), analyzed by ECIS. Analysis of TJP1 exon 20 inclusion by RT–PCR upon transfection of HUVECs with a control SSO or an SSO masking the exon 20–intron 20 boundary (E20 SSO) ( n = 9 independent biological replicates). Representative agarose gel on the right. Schematic outline at the top right. Endothelial resistance of control SSO‐ or E20 SSO‐transfected HUVECs ( n = 4 independent biological replicates), analyzed by ECIS. Data information: In (B–J), data are represented as mean ± SEM. n.s.: non‐significant, * P

    Techniques Used: In Vitro, Binding Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Over Expression, Electric Cell-substrate Impedance Sensing, Transfection, Agarose Gel Electrophoresis

    6) Product Images from "Real-Time Monitoring the Cytotoxic Effect of Andrographolide on Human Oral Epidermoid Carcinoma Cells"

    Article Title: Real-Time Monitoring the Cytotoxic Effect of Andrographolide on Human Oral Epidermoid Carcinoma Cells

    Journal: Biosensors

    doi: 10.3390/bios12050304

    ECIS measurement of OEC-M1 cell attachment and spreading. At time zero, OEC-M1 were inoculated in an ECIS, giving a final cell density of 1.25 × 10 5 cells per cm 2 . ( a ) Three-dimension representation of the electrical resistance as a function of frequency and time during the attachment and spreading of OEC-M1 cells on the sensing electrode. The red curve denotes the time-dependent resistance measured at 4 kHz. ( b ) Changes in resistance as a function of time measured at 4 kHz. Data were collected from eight independent electrodes for 24 h. These data are highly reproducible and similar to the red curve shown in ( a ). ( c ) Cells were inoculated into electrode-containing wells and allowed to develop into confluent layers for approximately 24 h. Andrographolide that was diluted in DMSO was then added to final concentrations of 10 μM (red), 30 μM (blue), 55 μM (green), 100 μM (yellow), and control (black), and the resultant changes in resistance were measured. Data were collected for 24 h after the addition of andrographolide. Only the resistance data at 4 kHz are shown.
    Figure Legend Snippet: ECIS measurement of OEC-M1 cell attachment and spreading. At time zero, OEC-M1 were inoculated in an ECIS, giving a final cell density of 1.25 × 10 5 cells per cm 2 . ( a ) Three-dimension representation of the electrical resistance as a function of frequency and time during the attachment and spreading of OEC-M1 cells on the sensing electrode. The red curve denotes the time-dependent resistance measured at 4 kHz. ( b ) Changes in resistance as a function of time measured at 4 kHz. Data were collected from eight independent electrodes for 24 h. These data are highly reproducible and similar to the red curve shown in ( a ). ( c ) Cells were inoculated into electrode-containing wells and allowed to develop into confluent layers for approximately 24 h. Andrographolide that was diluted in DMSO was then added to final concentrations of 10 μM (red), 30 μM (blue), 55 μM (green), 100 μM (yellow), and control (black), and the resultant changes in resistance were measured. Data were collected for 24 h after the addition of andrographolide. Only the resistance data at 4 kHz are shown.

    Techniques Used: Electric Cell-substrate Impedance Sensing, Cell Attachment Assay

    Effect of andrographolide on the wound healing migration of OEC-M1 cells, as measured by ECIS. Wound healing assay of andrographolide at concentrations of control (black), 10 (red), 30 (blue), 55 (green), and 100 µM (yellow) ( n = 5).
    Figure Legend Snippet: Effect of andrographolide on the wound healing migration of OEC-M1 cells, as measured by ECIS. Wound healing assay of andrographolide at concentrations of control (black), 10 (red), 30 (blue), 55 (green), and 100 µM (yellow) ( n = 5).

    Techniques Used: Migration, Electric Cell-substrate Impedance Sensing, Wound Healing Assay

    7) Product Images from "ECIS Based Electric Fence Method for Measurement of Human Keratinocyte Migration on Different Substrates"

    Article Title: ECIS Based Electric Fence Method for Measurement of Human Keratinocyte Migration on Different Substrates

    Journal: Biosensors

    doi: 10.3390/bios12050293

    Morphological change and micromotion analysis of HaCaT cells cultured on different substrates, which are control (bare array, black), collagen (red), PLL (blue), and PDL (green). ( a ) Morphology of HaCaT cells cultured on the electrodes with different coated substrates. Scale bar = 100 μm. ( b ) The averaged cell radius (r c ) is calculated by the morphological images using ImageJ software by the formula, r c = A c / π . ( c ) The resistance between cells (Rb) and ( d ) cell-substrate distance (h) are obtained by comparing the frequency scan data with the calculated values obtained from the cell-electrode model. Data were presented as mean ± SEM (N = 50 cells in each group for the analysis of cell radius, N > 5 in each group for the ECIS modeling analysis). * p
    Figure Legend Snippet: Morphological change and micromotion analysis of HaCaT cells cultured on different substrates, which are control (bare array, black), collagen (red), PLL (blue), and PDL (green). ( a ) Morphology of HaCaT cells cultured on the electrodes with different coated substrates. Scale bar = 100 μm. ( b ) The averaged cell radius (r c ) is calculated by the morphological images using ImageJ software by the formula, r c = A c / π . ( c ) The resistance between cells (Rb) and ( d ) cell-substrate distance (h) are obtained by comparing the frequency scan data with the calculated values obtained from the cell-electrode model. Data were presented as mean ± SEM (N = 50 cells in each group for the analysis of cell radius, N > 5 in each group for the ECIS modeling analysis). * p

    Techniques Used: Cell Culture, Software, Electric Cell-substrate Impedance Sensing

    The electric fence function (blue trace) prevents HaCaT cells from attaching and spreading on the ECIS electrode for 10 h. The black trace represents the simultaneous measurement without EF after seeding cells. EF is turned on at the cell inoculation and turned off after the cells form a confluent layer (magenta dashed line). Cells are inoculated at time = 0 h, and impedance measurements were performed by MFT using 11 pre-defined frequencies ranging from 62.5 Hz to 64 kHz. Here, only the resistance time-series data at 4 kHz are shown.
    Figure Legend Snippet: The electric fence function (blue trace) prevents HaCaT cells from attaching and spreading on the ECIS electrode for 10 h. The black trace represents the simultaneous measurement without EF after seeding cells. EF is turned on at the cell inoculation and turned off after the cells form a confluent layer (magenta dashed line). Cells are inoculated at time = 0 h, and impedance measurements were performed by MFT using 11 pre-defined frequencies ranging from 62.5 Hz to 64 kHz. Here, only the resistance time-series data at 4 kHz are shown.

    Techniques Used: Electric Cell-substrate Impedance Sensing

    ( a ) Schematic of the ECIS system. ( b ) Image of the ECIS probe station connected with two electrode arrays. Each array contains eight rectangular electrode wells. ( c ) For an 8W1E array, each of the eight wells has one sensing electrode with 250-μm in diameter. ( d ) For an 8W10E array, each of the eight wells has ten identical sensing electrodes with 250-μm in diameter.
    Figure Legend Snippet: ( a ) Schematic of the ECIS system. ( b ) Image of the ECIS probe station connected with two electrode arrays. Each array contains eight rectangular electrode wells. ( c ) For an 8W1E array, each of the eight wells has one sensing electrode with 250-μm in diameter. ( d ) For an 8W10E array, each of the eight wells has ten identical sensing electrodes with 250-μm in diameter.

    Techniques Used: Electric Cell-substrate Impedance Sensing

    Initial adhesion analysis of HaCaT cells seeded on different substrates. ( a ) Relative cell initial adhesion on different coated substrates, which are control (bare array, black), collagen (red), PLL (blue), and PDL (green), via alamarBlue ® cell viability assay. ( b ) Diagrams of the cell adhesion process and the capacitance profile in the time series data measured by ECIS. ( c ) Analysis of HaCaT cell adhesion time on different coated substrates using the ECIS time-course measurement. Data were presented as mean ± SEM (N > 8 for the alamarBlue ® cell viability assay, N > 10 for the analysis of cell adhesion time, and the N number is the total wells). * p
    Figure Legend Snippet: Initial adhesion analysis of HaCaT cells seeded on different substrates. ( a ) Relative cell initial adhesion on different coated substrates, which are control (bare array, black), collagen (red), PLL (blue), and PDL (green), via alamarBlue ® cell viability assay. ( b ) Diagrams of the cell adhesion process and the capacitance profile in the time series data measured by ECIS. ( c ) Analysis of HaCaT cell adhesion time on different coated substrates using the ECIS time-course measurement. Data were presented as mean ± SEM (N > 8 for the alamarBlue ® cell viability assay, N > 10 for the analysis of cell adhesion time, and the N number is the total wells). * p

    Techniques Used: Viability Assay, Electric Cell-substrate Impedance Sensing

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    Applied BioPhysics ecis model zθ
    Role of Junctions in Opto-RhoGEF induced changes in vascular barrier strength. ( A )Linearity Index for a BOEC monolayer stably expressing Lck-mTurquoise2-iLID and either SspB-HaloTag-TIAM1(DHPH) (purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) for the frame before photo activation (pre) and the last frame of photo activation (Activation). Images of the junctions are in Fig. S2.2 and example of linearity index analysis in Fig. S3.1A . Each dot represents and individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The number of cells is: Opto-TIAM pre=55, Opto-TIAM Activation=52, Opto-ITSN pre=71, Opto-ITSN Activation=76, Opto-P63 pre=74, Opto-P63 Activation=51. The data is from two independent experiments. ( B ) Resistance of a monolayer of BOECs stably expressing Lck-mTurquoise2-iLID, solely as a control (grey), and either SspB-HaloTag-TIAM1(DHPH)(purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) measured with <t>ECIS</t> at 4000 Hz every 10 s. Cyan bars indicated photo activation with blue LED light (60 min, 15 min). Gray bar with dashed lines indicates the addition of VE-cadherin blocking antibody in medium (darker color line) or medium as a control (lighter color line). At the end of the gray bar the medium is replaced for all conditions. Thin lines represent the average value from one well of an 8W10E PET ECIS array. Thick lines represent the mean. Four wells were measured for each condition. ( C ) Representative zoom ins from confocal microscopy images of a BOEC monolayer stably expressing Lck-mTurquoise2-iLID (not shown) and either SspB-HaloTag-TIAM1(DHPH)/ ITSN1(DHPH) or p63RhoGEF(DH) stained with JF552 nm dye (LUT = mpl-magma, bright colors indicating higher intensity). Additionally, stained for VE-Cadherin with the live labeling antibody Alexa Fluor 647 Mouse Anti-Human CD144 (white in merge, grey inverted in single channel). Left panel shows untreated cells, right panel shows cells treated with the VE-cadherin blocking antibody. Scale bars: 25 µm. Times are min:s from the start of the recording. Gray bar indicates the condition before photo activation. Cyan bar indicates 442 nm photo activation. Arrows indicate overlap and protrusions. Asterisks indicate holes in monolayer.
    Ecis Model Zθ, supplied by Applied BioPhysics, used in various techniques. Bioz Stars score: 96/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Role of Junctions in Opto-RhoGEF induced changes in vascular barrier strength. ( A )Linearity Index for a BOEC monolayer stably expressing Lck-mTurquoise2-iLID and either SspB-HaloTag-TIAM1(DHPH) (purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) for the frame before photo activation (pre) and the last frame of photo activation (Activation). Images of the junctions are in Fig. S2.2 and example of linearity index analysis in Fig. S3.1A . Each dot represents and individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The number of cells is: Opto-TIAM pre=55, Opto-TIAM Activation=52, Opto-ITSN pre=71, Opto-ITSN Activation=76, Opto-P63 pre=74, Opto-P63 Activation=51. The data is from two independent experiments. ( B ) Resistance of a monolayer of BOECs stably expressing Lck-mTurquoise2-iLID, solely as a control (grey), and either SspB-HaloTag-TIAM1(DHPH)(purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) measured with ECIS at 4000 Hz every 10 s. Cyan bars indicated photo activation with blue LED light (60 min, 15 min). Gray bar with dashed lines indicates the addition of VE-cadherin blocking antibody in medium (darker color line) or medium as a control (lighter color line). At the end of the gray bar the medium is replaced for all conditions. Thin lines represent the average value from one well of an 8W10E PET ECIS array. Thick lines represent the mean. Four wells were measured for each condition. ( C ) Representative zoom ins from confocal microscopy images of a BOEC monolayer stably expressing Lck-mTurquoise2-iLID (not shown) and either SspB-HaloTag-TIAM1(DHPH)/ ITSN1(DHPH) or p63RhoGEF(DH) stained with JF552 nm dye (LUT = mpl-magma, bright colors indicating higher intensity). Additionally, stained for VE-Cadherin with the live labeling antibody Alexa Fluor 647 Mouse Anti-Human CD144 (white in merge, grey inverted in single channel). Left panel shows untreated cells, right panel shows cells treated with the VE-cadherin blocking antibody. Scale bars: 25 µm. Times are min:s from the start of the recording. Gray bar indicates the condition before photo activation. Cyan bar indicates 442 nm photo activation. Arrows indicate overlap and protrusions. Asterisks indicate holes in monolayer.

    Journal: bioRxiv

    Article Title: Opto-RhoGEFs: an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength

    doi: 10.1101/2022.10.17.512253

    Figure Lengend Snippet: Role of Junctions in Opto-RhoGEF induced changes in vascular barrier strength. ( A )Linearity Index for a BOEC monolayer stably expressing Lck-mTurquoise2-iLID and either SspB-HaloTag-TIAM1(DHPH) (purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) for the frame before photo activation (pre) and the last frame of photo activation (Activation). Images of the junctions are in Fig. S2.2 and example of linearity index analysis in Fig. S3.1A . Each dot represents and individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The number of cells is: Opto-TIAM pre=55, Opto-TIAM Activation=52, Opto-ITSN pre=71, Opto-ITSN Activation=76, Opto-P63 pre=74, Opto-P63 Activation=51. The data is from two independent experiments. ( B ) Resistance of a monolayer of BOECs stably expressing Lck-mTurquoise2-iLID, solely as a control (grey), and either SspB-HaloTag-TIAM1(DHPH)(purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) measured with ECIS at 4000 Hz every 10 s. Cyan bars indicated photo activation with blue LED light (60 min, 15 min). Gray bar with dashed lines indicates the addition of VE-cadherin blocking antibody in medium (darker color line) or medium as a control (lighter color line). At the end of the gray bar the medium is replaced for all conditions. Thin lines represent the average value from one well of an 8W10E PET ECIS array. Thick lines represent the mean. Four wells were measured for each condition. ( C ) Representative zoom ins from confocal microscopy images of a BOEC monolayer stably expressing Lck-mTurquoise2-iLID (not shown) and either SspB-HaloTag-TIAM1(DHPH)/ ITSN1(DHPH) or p63RhoGEF(DH) stained with JF552 nm dye (LUT = mpl-magma, bright colors indicating higher intensity). Additionally, stained for VE-Cadherin with the live labeling antibody Alexa Fluor 647 Mouse Anti-Human CD144 (white in merge, grey inverted in single channel). Left panel shows untreated cells, right panel shows cells treated with the VE-cadherin blocking antibody. Scale bars: 25 µm. Times are min:s from the start of the recording. Gray bar indicates the condition before photo activation. Cyan bar indicates 442 nm photo activation. Arrows indicate overlap and protrusions. Asterisks indicate holes in monolayer.

    Article Snippet: Resistance Measurement in an Endothelial Monolayer To perform resistance measurements, the ECIS® ZTheta (Applied BioPhysics) machine was used.

    Techniques: Stable Transfection, Expressing, Activation Assay, Electric Cell-substrate Impedance Sensing, Blocking Assay, Positron Emission Tomography, Confocal Microscopy, Staining, Labeling

    Photoactivation of Opto-RhoGEFs controls permeability and vascular barrier strength. ( A ) Fluorescence intensity measured in a transwell assay for a monolayer of BOECs stably expressing Lck-mTurquoise2-iLID, solely as a control, and SspB-HaloTag-TIAM1(DHPH) treated with FITC 0.3 kDa, FITC dextran 10 kDa and FITC dextran 70 kDa. Photo activated with blue LED light for 10 min as indicated by cyan background in the graph or kept in the dark indicated by white background. Dots represent individual transwell dishes. The black bar indicates the mean. The number of transwell dishes per condition is: Control_Lck-iLID=9, OpotTIAM_dark=6, OptoTIAM_light=9. The data is from three experiments. ( B ) Resistance of a monolayer of BOECs stably expressing Lck-mTurquoise2-iLID, solely as a control (grey), and either SspB-HaloTag-TIAM1(DHPH)(purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) measured with ECIS at 4000 Hz every 10 s. Cyan bars indicated photo activation with blue LED light (1 min, 5 min, 10 min, 3x 15 min, 120 min). Thin lines represent the average value from one well of an 8W10E PET ECIS array. Thick lines represent the mean. The number of wells per condition was: OptoTIAM=6, OptoITSN=6, OptoP63=6, control=4. ( C ) Representative zoom ins from confocal microscopy images of a BOEC monolayer stably expressing Lck-mTurquoise2-iLID (not shown) and either SspB-HaloTag-TIAM1(DHPH)/ ITSN1(DHPH) or p63RhoGEF(DH) stained with JF552 nm dye (LUT = mpl-magma, bright colors indicating higher intensity). Additionally, stained for VE-Cadherin with the live labeling antibody Alexa Fluor 647 Mouse Anti-Human CD144 (white). Scale bars: 25 µm. Times are min:s from the start of the recording. Cyan bar indicates 442 nm photo activation. Arrows indicate overlap and protrusions. Asterisks indicate holes in monolayer. Whole field of view is shown in S4.2 .

    Journal: bioRxiv

    Article Title: Opto-RhoGEFs: an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength

    doi: 10.1101/2022.10.17.512253

    Figure Lengend Snippet: Photoactivation of Opto-RhoGEFs controls permeability and vascular barrier strength. ( A ) Fluorescence intensity measured in a transwell assay for a monolayer of BOECs stably expressing Lck-mTurquoise2-iLID, solely as a control, and SspB-HaloTag-TIAM1(DHPH) treated with FITC 0.3 kDa, FITC dextran 10 kDa and FITC dextran 70 kDa. Photo activated with blue LED light for 10 min as indicated by cyan background in the graph or kept in the dark indicated by white background. Dots represent individual transwell dishes. The black bar indicates the mean. The number of transwell dishes per condition is: Control_Lck-iLID=9, OpotTIAM_dark=6, OptoTIAM_light=9. The data is from three experiments. ( B ) Resistance of a monolayer of BOECs stably expressing Lck-mTurquoise2-iLID, solely as a control (grey), and either SspB-HaloTag-TIAM1(DHPH)(purple)/ ITSN1(DHPH) (blue) or p63RhoGEF(DH) (green) measured with ECIS at 4000 Hz every 10 s. Cyan bars indicated photo activation with blue LED light (1 min, 5 min, 10 min, 3x 15 min, 120 min). Thin lines represent the average value from one well of an 8W10E PET ECIS array. Thick lines represent the mean. The number of wells per condition was: OptoTIAM=6, OptoITSN=6, OptoP63=6, control=4. ( C ) Representative zoom ins from confocal microscopy images of a BOEC monolayer stably expressing Lck-mTurquoise2-iLID (not shown) and either SspB-HaloTag-TIAM1(DHPH)/ ITSN1(DHPH) or p63RhoGEF(DH) stained with JF552 nm dye (LUT = mpl-magma, bright colors indicating higher intensity). Additionally, stained for VE-Cadherin with the live labeling antibody Alexa Fluor 647 Mouse Anti-Human CD144 (white). Scale bars: 25 µm. Times are min:s from the start of the recording. Cyan bar indicates 442 nm photo activation. Arrows indicate overlap and protrusions. Asterisks indicate holes in monolayer. Whole field of view is shown in S4.2 .

    Article Snippet: Resistance Measurement in an Endothelial Monolayer To perform resistance measurements, the ECIS® ZTheta (Applied BioPhysics) machine was used.

    Techniques: Permeability, Fluorescence, Transwell Assay, Stable Transfection, Expressing, Electric Cell-substrate Impedance Sensing, Activation Assay, Positron Emission Tomography, Confocal Microscopy, Staining, Labeling

    TUG1 is not important for basal cell turnover, barrier or mitochondrial function, migration and monocyte adhesion. (A)–(G) HUVECs were transfected with two LNA GapmeRs against TUG1 —LNA TUG1_1 and LNA TUG1_2 –and LNA Ctrl (10 nM) and (A) expression levels were measured after 48 hours by RT-qPCR. Expression is relative to GAPDH (n = 4; SEM; RM one-way ANOVA with Greenhouse-Geisser correction and Sidak multiple comparison test). (B) Relative cell growth determined from cell count at 0 h, 24 h, 48 h and 72 h (n = 3; SEM; RM Two-way ANOVA with Tuckey multiple comparison test). (C) Caspase-3/7 activity was measured by determination of fluorescence with ELISA plate reader (n = 3; SEM; One-way ANOVA with Holm-Sidak correction). Staurosporine was taken along as a postive control. (D) Cell-cell interactions (Rb) and cell-matrix-interactions (α) were measured by Electric Cell Impedance Sensing (ECIS; n = 3; SEM; Kruskal-Wallis-test with Dunn´s correction). (E) Determination of re-establishment of monolayer after wounding using ECIS (n = 3; SEM; One-way ANOVA with Holm-Sidak multiple comparison test). (F) Seahorse mitochondrial stress test assessing multiple mitochondrial characteristics via measurement of changes in Oxygen Consumption Rate (OCR) after serial injection of Oligomycin, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) and Rotenone A/Antimycin (n = 3; SEM; One-way ANOVA with Holm-Sidak multiple comparison test. One representative experiment displaying the changes of OCR throughout the progress of the Seahorse mitochondrial stress test assay. (G) Assessment of monocyte adhesion with and without TNF-α stimulation. (n = 3; SEM; Two-way ANOVA with Tuckey multiple comparison test).

    Journal: PLoS ONE

    Article Title: Aging-regulated TUG1 is dispensable for endothelial cell function

    doi: 10.1371/journal.pone.0265160

    Figure Lengend Snippet: TUG1 is not important for basal cell turnover, barrier or mitochondrial function, migration and monocyte adhesion. (A)–(G) HUVECs were transfected with two LNA GapmeRs against TUG1 —LNA TUG1_1 and LNA TUG1_2 –and LNA Ctrl (10 nM) and (A) expression levels were measured after 48 hours by RT-qPCR. Expression is relative to GAPDH (n = 4; SEM; RM one-way ANOVA with Greenhouse-Geisser correction and Sidak multiple comparison test). (B) Relative cell growth determined from cell count at 0 h, 24 h, 48 h and 72 h (n = 3; SEM; RM Two-way ANOVA with Tuckey multiple comparison test). (C) Caspase-3/7 activity was measured by determination of fluorescence with ELISA plate reader (n = 3; SEM; One-way ANOVA with Holm-Sidak correction). Staurosporine was taken along as a postive control. (D) Cell-cell interactions (Rb) and cell-matrix-interactions (α) were measured by Electric Cell Impedance Sensing (ECIS; n = 3; SEM; Kruskal-Wallis-test with Dunn´s correction). (E) Determination of re-establishment of monolayer after wounding using ECIS (n = 3; SEM; One-way ANOVA with Holm-Sidak multiple comparison test). (F) Seahorse mitochondrial stress test assessing multiple mitochondrial characteristics via measurement of changes in Oxygen Consumption Rate (OCR) after serial injection of Oligomycin, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) and Rotenone A/Antimycin (n = 3; SEM; One-way ANOVA with Holm-Sidak multiple comparison test. One representative experiment displaying the changes of OCR throughout the progress of the Seahorse mitochondrial stress test assay. (G) Assessment of monocyte adhesion with and without TNF-α stimulation. (n = 3; SEM; Two-way ANOVA with Tuckey multiple comparison test).

    Article Snippet: The resulting potential was detected by the ECIS instrument Zθ (Applied BioPhysics).

    Techniques: Migration, Transfection, Expressing, Quantitative RT-PCR, Cell Counting, Activity Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Electric Cell-substrate Impedance Sensing, Injection