Journal: PLoS ONE

Article Title: DNA Repair and Cell Cycle Biomarkers of Radiation Exposure and Inflammation Stress in Human Blood

doi: 10.1371/journal.pone.0048619

Figure Lengend Snippet: Protein levels of phosphorylated CHK2-thr68 in protein lysate from cultured whole blood in the presence or absence of LPS (50 ng/ml) were measured by ELISA. Absorbance values were normalized with respect to the average pCHK2-thr68 level in unirradiated donors. In the absence of LPS, radiation induced CHK2-thr68 levels ∼1.6-fold (±0.1) relative to sham irradiated samples, whereas in the presence of LPS, CHK2-thr68 levels were indistinguishable from sham irradiated samples (p>0.4).

Article Snippet: Amounts of protein in lysate or plasma were quantified using ELISA kits; human BAX ELISA kit (Assay Designs), human phosphorylated CHK2-thr68 ELISA kit (Cell Signalling).

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

Journal: PLoS ONE

Article Title: DNA Repair and Cell Cycle Biomarkers of Radiation Exposure and Inflammation Stress in Human Blood

doi: 10.1371/journal.pone.0048619

Figure Lengend Snippet: In comparison to untreated sham samples, inflammation in the absence of radiation exposure upregulates CDKN1A (red) and downregulates FDXR and BBC3 (green). Samples exposed to 2 Gy radiation only exhibit increased expression of all nine biomarkers, whereas subjects exposed to 2 Gy plus inflammation stress show modified induction of CDKN1A , FDXR and BBC3 and abrogation of the phosphorylation of CHK2 protein. The arrows in the radiation and inflammation combined treatment group indicate the direction of expression relative to the radiation alone group.

Article Snippet: Amounts of protein in lysate or plasma were quantified using ELISA kits; human BAX ELISA kit (Assay Designs), human phosphorylated CHK2-thr68 ELISA kit (Cell Signalling).

Techniques: Expressing, Modification

Journal: PLoS ONE

Article Title: DNA Repair and Cell Cycle Biomarkers of Radiation Exposure and Inflammation Stress in Human Blood

doi: 10.1371/journal.pone.0048619

Figure Lengend Snippet: Classification accuracy based on ten 10-fold cross-validation as a function of the number of markers considered, based on order determined during filtering with the Gini index. The four classes used in this analysis are: radiation only (R), inflammation stress only (L), combined exposures involving both radiation and LPS (RL), and samples with no radiation exposure and no LPS treatment (N). Marker order is: PCNA, CDKN1A, pCHK2-thr68, BBC3, FDXR, DDB2, XPC, POLH, and GADD45a. Maximum classification accuracy was 0.88 for the top 5-marker set.

Article Snippet: Amounts of protein in lysate or plasma were quantified using ELISA kits; human BAX ELISA kit (Assay Designs), human phosphorylated CHK2-thr68 ELISA kit (Cell Signalling).

Techniques: Marker

Journal: BioMed Research International

Article Title: STAT6 siRNA Matrix-Loaded Gelatin Nanocarriers: Formulation, Characterization, and Ex Vivo Proof of Concept Using Adenocarcinoma Cells

doi: 10.1155/2013/858946

Figure Lengend Snippet: Preparation of S6S-GNC. The 1% w/v aqueous gelatin solution is incubated with the STAT6 siRNA for 10 min at 35°C, and then ethanol and crosslinker are added dropwise at a stirring rate of 600 rpm at 35°C for 1 hr, at which point the stirring rate is reduced to 200 rpm. After approximately 4 h, the ethanol is completely evaporated, and STAT6 siRNA loaded gelatin nanocarriers remain in a colloidal suspension in water or PBS pH 7.4. The resultant nanoparticles were collected by centrifugation and resuspended for subsequent characterization or lyophilization in the presence of 1% w/w lactose monohydrate.

Article Snippet: Human STAT6 and β -Actin proteins (Cell Signaling, USA) were detected using rabbit polyclonal primary antibodies (Santa Cruz Biotechnology, Inc., USA).

Techniques: Incubation, Centrifugation

Journal: BioMed Research International

Article Title: STAT6 siRNA Matrix-Loaded Gelatin Nanocarriers: Formulation, Characterization, and Ex Vivo Proof of Concept Using Adenocarcinoma Cells

doi: 10.1155/2013/858946

Figure Lengend Snippet: In vitro STAT6 siRNA release profile for the S6S-GNC formulation compared to the STAT6 siRNA solution. Lyophilized formulation was resuspended in PBS pH 7.4 and filled inside dialysis membrane bags with MWCO of 300 kDa (Sigma, USA). The membrane bags were placed in 50 mL of PBS medium maintained at a temperature of 37 ± 2°C with continuous gentle stirring at 300 rpm on a magnetic heating and stirring plate. At specific time intervals, 0.5 mL aliquots of dissolution medium were withdrawn and analyzed using a Biospek UV spectrophotometer. Results are represented as mean ± standard deviations (where n = 3).

Article Snippet: Human STAT6 and β -Actin proteins (Cell Signaling, USA) were detected using rabbit polyclonal primary antibodies (Santa Cruz Biotechnology, Inc., USA).

Techniques: In Vitro, Spectrophotometry

Journal: BioMed Research International

Article Title: STAT6 siRNA Matrix-Loaded Gelatin Nanocarriers: Formulation, Characterization, and Ex Vivo Proof of Concept Using Adenocarcinoma Cells

doi: 10.1155/2013/858946

Figure Lengend Snippet: Measurement of STAT6 protein expression by western blot. The effect of STAT6 siRNA-GNC on the expression of STAT6 in A549-treated lung cancer cells was shown. A549 cells were preincubated with S6S-GNC and S6S-lipofectamine complex and without any treatment (control). The cells were lysed, and STAT6 protein expression was analyzed by western blot of whole cell lysates. The β -Actin expression was analyzed as a loading control.

Article Snippet: Human STAT6 and β -Actin proteins (Cell Signaling, USA) were detected using rabbit polyclonal primary antibodies (Santa Cruz Biotechnology, Inc., USA).

Techniques: Expressing, Western Blot

Journal: Scientific Reports

Article Title: Spatial and temporal VEGF receptor intracellular trafficking in microvascular and macrovascular endothelial cells

doi: 10.1038/s41598-021-96964-7

Figure Lengend Snippet: VEGF-induced VEGFR migration to the nucleus in micro- and macrovascular endothelial cells . The nuclear and cytoplasmic levels of VEGFR1 and VEGFR2 in HREC and HAEC were evaluated by immunofluorescence plus sandwich ELISA at different time points (10 min, 30 min and 2 h) after exposure to 100 ng/ml VEGF and compared to saline controls (NO-VEGF). Representative 3D reconstruction rendering images of the HREC and HAEC after VEGF treatment and control immunostained for ( A ) C-terminal VEGFR1 and VEGFR2. The top image of each block is the horizontal section of the 3D rendering below used as a way of showing the amount of the respective receptor inside the nucleus. Scale bars = 3 μm, and ( D ) N-terminal VEGFR1 and VEGFR2. The top left image of each block shows the position of the VEGFR1 nuclear translocation in only one focal plane and the correlation of this point are located in the 3D lateral and lower cross sections. Scale bars = 5 μm. ( B and E ) Graphs showing the quantification of the median intensity of fluorescence (MIF) volume of at least 10 confocal z-stack images at 400 × magnification for each experiment performed using NIS elements software. ( C and F ) Graphs showing the quantification of the total VEGFR1 and VEGFR2 present in nuclear and cytoplasmic fractions of the HREC and HAEC after VEGF treatment and control quantified by sandwich ELISA. All graphs represent VEGF time point fold change with respect to control NO-VEGF and the graphs bars represents the average of the a minimum of 3 separate experiments from 3 donors of the HREC and 2 donors of the HAEC. * = p < 0.05 as determined by 1-way ANOVA and multi-comparison post-hoc Tukey’s test was considered significant.

Article Snippet: Total VEGFR levels were quantified using Quantikine sandwich ELISA for Human VEGFR1 (#DVR100C R&D) and PathScan sandwich ELISA for Human VEGFR2 (#7340 Cell signaling) and phosphorylated VEGFRs were quantified using DuoSet IC sandwich ELISA for Human Phospho-VEGFR1 (#DYC4170 R&D) and PathScan sandwich ELISA for Human Phospho-Y1175-VEGFR2 (#7335 Cell signaling) according to the manufacturer’s instructions.

Techniques: Migration, Immunofluorescence, Sandwich ELISA, Blocking Assay, Translocation Assay, Fluorescence, Software

Journal: Scientific Reports

Article Title: Spatial and temporal VEGF receptor intracellular trafficking in microvascular and macrovascular endothelial cells

doi: 10.1038/s41598-021-96964-7

Figure Lengend Snippet: Intravitreal injection of VEGF promotes nuclear localization of VEGFR1 and VEGFR2 in mouse microavascular retinal endothelial cells . Electron microscopy (EM) of VEGFR1 and VEGFR2 immunogold staining in C57BL/6 J mouse retinal endothelial cells. The nuclear and cytoplasmic levels of VEGFRs in retinal endothelial cells from C57BL/6 J mice were evaluated at different time points (2, 6 and 24 h – n = 3/time point) after intravitreal injection of 10 ng VEGF. Negative controls were either: a) untreated eyes or b) eyes that received intravitreal injection of the vehicle (1 ul of the 0.9% saline). ( A ) Double immunogold staining was achieved using two different size of the gold particles to differentiate VEGFR1 (10 nm gold) from VEGFR2 (6 nm). ( B ) Representative EM images from retinal endothelial cells from a control unstimulated eye. ( C ) Negative control showing no specific gold particles in a retinal endothelial cell when the primary antibody was omitted. ( D–E ) Representative EM images from retinal endothelial cells from 2 and 24 h VEGF-stimulated eyes showing expression of VEFR1 (large arrows) and VEGFR2 (small arrows). Scale bar = 200 nm. Graphs show the quantification of the number of gold particles per area (5 µm 2 ) in the nucleus ( F and G ) and cytoplasm ( I and J ) of at least 10 endothelial cells at 11,000 × magnification for each experiment performed using ImageJ software. ( H and K ) The ratio of VEGFR1:VEGFR2 in the nucleus and cytoplasm. Experiments was repeated 2 times for each animal. * = p < 0.05 as determined by 1-way ANOVA and multi-comparison post-hoc Tukey’s test is considered significant.

Article Snippet: Total VEGFR levels were quantified using Quantikine sandwich ELISA for Human VEGFR1 (#DVR100C R&D) and PathScan sandwich ELISA for Human VEGFR2 (#7340 Cell signaling) and phosphorylated VEGFRs were quantified using DuoSet IC sandwich ELISA for Human Phospho-VEGFR1 (#DYC4170 R&D) and PathScan sandwich ELISA for Human Phospho-Y1175-VEGFR2 (#7335 Cell signaling) according to the manufacturer’s instructions.

Techniques: Injection, Electron Microscopy, Staining, Negative Control, Expressing, Software

Journal: Scientific Reports

Article Title: Spatial and temporal VEGF receptor intracellular trafficking in microvascular and macrovascular endothelial cells

doi: 10.1038/s41598-021-96964-7

Figure Lengend Snippet: The nuclear VEGFR1:VEGFR2 ratio is different in micro- and macrovascular endothelial cells and VEGF-stimulated nuclear VEGFRs are minimally phosphorylated . The nuclear levels of VEGFR1 and VEGFR2 plus of phospho-VEGFR1 and phospho-VEGFR2 in HREC and HAEC were evaluated by double immunofluorescence and sandwich ELISA, respectively, at different time points (10 min, 30 min and 2 h) after exposure to 100 ng/ml VEGF and compared to untreated controls (NO-VEGF). ( A ) Representative 3D reconstruction rendering images of the HREC and HAEC after VEGF treatment and control immunostained for full length VEGFR1 (red) + C-terminal VEGFR2 (green) showing simultaneous nuclear localization of both receptors after VEGF treatment. The nuclei were fluorescently stained with the DNA intercalating dye Hoechst (blue) and the blue channel was partially removed from the 3D representations to evidence the simultaneous localization and association of VEGFR1 and VEGFR2 in the area corresponding to the nucleus. The associated points that spatial colocalize green and red are shown in yellow. Scale bars = 5 μm. ( B ) Graphic representation of the nuclear VEGFR1:VEGFR2 ratio of HREC and HAEC following addition of VEGF shown in A after the quantification of the median intensity of fluorescence (MIF) of at least 10 confocal z-stack images at 400 × magnification for each experiment performed using NIS elements software. VEGFR1:VEGFR2 ratio graphs represent VEGF time point fold change with respect to control NO-VEGF and the graphs bars represents the average of the a minimum of 3 separate experiments from HREC and HAEC. Graphs showing the quantification of the phospho-VEGFR1 ( C–D , G–H) and phospho-VEGFR2 ( K–L , O–P ) present in nuclear ( C , G , K and O ) and cytoplasmic ( D, H, L and P ) fractions of the HREC and HAEC after VEGF treatment and control quantified by sandwich ELISA. ( E–F , I–J , M–N , Q–R ) graphic representation of the phospho-VEGFR1 and phospho-VEGFR2 percentage relative to total amount of VEGFR1 or VEGFR2 found in the nucleus and cytoplasm of the HREC and HAEC following addition of VEGF. ELISA data for each VEGF time point was normalized by the control NO-VEGF and bars represent the average of the a minimum of 3 separate experiments from 3 donors of the HREC and 2 donors of the HAEC. * = p < 0.05 as determined by 1-way ANOVA and multi-comparison post-hoc Tukey’s test was considered significant.

Article Snippet: Total VEGFR levels were quantified using Quantikine sandwich ELISA for Human VEGFR1 (#DVR100C R&D) and PathScan sandwich ELISA for Human VEGFR2 (#7340 Cell signaling) and phosphorylated VEGFRs were quantified using DuoSet IC sandwich ELISA for Human Phospho-VEGFR1 (#DYC4170 R&D) and PathScan sandwich ELISA for Human Phospho-Y1175-VEGFR2 (#7335 Cell signaling) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Sandwich ELISA, Staining, Fluorescence, Software, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Spatial and temporal VEGF receptor intracellular trafficking in microvascular and macrovascular endothelial cells

doi: 10.1038/s41598-021-96964-7

Figure Lengend Snippet: VEGFR2 can translocate from plasma membrane and intracellular compartments to the nucleus via early endosomes following VEGF treatment in endothelial cells . Intracellular trafficking of VEGFR2 in HREC and HAEC was evaluated by immunofluorescence at different time points (10 min, 30 min and 2 h) after exposure to 100 ng/ml VEGF and compared to saline controls (NO-VEGF). Triple immunofluorescence was performed to colocalize VEGFR2 with specific organelles. ( A ) 3D reconstruction rendering of the HREC and HAEC immunostained with VEGFR2 (green), trans Golgi (red) and calreticulin (endoplasmic reticulum-ER gray) ( B ) 3D reconstruction rendering of HREC and HAEC immunostained with VEGFR2 (green), LAMP1 (lysosome-red) and EEA1 (early endosome-magenta). The nuclei in ( A ) and ( B ) were fluorescently stained with the DNA intercalating dye Hoechst (blue). The colocalization points between green and red are shown in yellow and between green and magenta shown in gray. The top image of each block shows the 3D rendering of the representative cells of each time point and the same cells are shown in the lower image of each block showing the position of the nuclear translocation of VEGFR2 in only one focal plane in the center and the correlation of this point located in the 3D lateral and lower cross sections. Scale bars = 5 μm. ( C ) Quantification of the number of VEGFR2 colocalized voxels with the organelles relative to the number of cells in each image. The results show the analyzes of at least 10 confocal z-stack images at 400 × magnification for each time point by Imaris software and summarize the data obtained by quantifying the organelles from a minimum of 3 separate experiments from 3 donors of the HREC and 2 donors of the HAEC. The results show that the colocalization of VEGFR2 with trans Golgi and early endosomes increases between 10 and 30 min after VEGF treatment compared to control and correlates with the increase of VEGFR2 in the nucleus in HREC, but not in HAEC. * = p < 0.05 as determined by 1-way ANOVA and multi-comparison post-hoc Tukey’s test is considered significant.

Article Snippet: Total VEGFR levels were quantified using Quantikine sandwich ELISA for Human VEGFR1 (#DVR100C R&D) and PathScan sandwich ELISA for Human VEGFR2 (#7340 Cell signaling) and phosphorylated VEGFRs were quantified using DuoSet IC sandwich ELISA for Human Phospho-VEGFR1 (#DYC4170 R&D) and PathScan sandwich ELISA for Human Phospho-Y1175-VEGFR2 (#7335 Cell signaling) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Blocking Assay, Translocation Assay, Software

Journal: Scientific Reports

Article Title: Spatial and temporal VEGF receptor intracellular trafficking in microvascular and macrovascular endothelial cells

doi: 10.1038/s41598-021-96964-7

Figure Lengend Snippet: Depletion of the biosynthetic pool by Brefeldin A prevents nuclear translocation of VEGFR2 into VEGF-stimulated microvascular endothelial cells . Intracellular trafficking of VEGFR1 and VEGFR2 in HREC and HAEC was evaluated by immunofluorescence at different time points (10 min, 30 min and 2 h) after exposure to 100 ng/ml VEGF and compared to saline controls (NO-VEGF). Triple immunofluorescence was performed to confirm the biosynthetic pool depletion after Brefeldin A treatment and evaluate the nuclear translocation of the VEGFRs by measuring the MIF. ( A–B ) 3D reconstruction rendering of the HREC and HAEC immunostained with VEGFR1 and VEGFR2 (green), respectively, plus trans Golgi (red), calreticulin (endoplasmic reticulum-ER gray) and DNA intercalating dye Hoechst (nuclei-blue). The colocalization points between green and red are shown in yellow and between green and gray shown in cyan. Scale bars = 10 μm. ( C ) Graphs showing the quantification of the median intensity of fluorescence (MIF) volume of at least 10 confocal z-stack images at 400 × magnification for each experiment performed using NIS elements software. MIF graphs represent VEGF time point fold change with respect to control vehicle treated NO-VEGF and the graphs bars represents the average of the a minimum of 3 separate experiments from HREC and HAEC. The results show that the nuclear translocating pool of the VEGFR2 in HREC are derived from the Golgi. * = p < 0.05 as determined by 2-way ANOVA followed by multi-comparison post-hoc Tukey’s test shows intra-group differences with respect to the VEGF treatment and # = p < 0.05 2-way ANOVA followed by multi-comparison post-hoc Tukey’s and t-student tests shows inter-group differences with respect to the Vehicle and Brefeldin A treatment.

Article Snippet: Total VEGFR levels were quantified using Quantikine sandwich ELISA for Human VEGFR1 (#DVR100C R&D) and PathScan sandwich ELISA for Human VEGFR2 (#7340 Cell signaling) and phosphorylated VEGFRs were quantified using DuoSet IC sandwich ELISA for Human Phospho-VEGFR1 (#DYC4170 R&D) and PathScan sandwich ELISA for Human Phospho-Y1175-VEGFR2 (#7335 Cell signaling) according to the manufacturer’s instructions.

Techniques: Translocation Assay, Immunofluorescence, Fluorescence, Software, Derivative Assay

Journal: Scientific Reports

Article Title: Spatial and temporal VEGF receptor intracellular trafficking in microvascular and macrovascular endothelial cells

doi: 10.1038/s41598-021-96964-7

Figure Lengend Snippet: VEGF-induced VEGFR migration to the nucleus in micro- and macrovascular endothelial cells . The nuclear and cytoplasmic levels of VEGFR1 and VEGFR2 in HREC and HAEC were evaluated by immunofluorescence plus sandwich ELISA at different time points (10 min, 30 min and 2 h) after exposure to 100 ng/ml VEGF and compared to saline controls (NO-VEGF). Representative 3D reconstruction rendering images of the HREC and HAEC after VEGF treatment and control immunostained for ( A ) C-terminal VEGFR1 and VEGFR2. The top image of each block is the horizontal section of the 3D rendering below used as a way of showing the amount of the respective receptor inside the nucleus. Scale bars = 3 μm, and ( D ) N-terminal VEGFR1 and VEGFR2. The top left image of each block shows the position of the VEGFR1 nuclear translocation in only one focal plane and the correlation of this point are located in the 3D lateral and lower cross sections. Scale bars = 5 μm. ( B and E ) Graphs showing the quantification of the median intensity of fluorescence (MIF) volume of at least 10 confocal z-stack images at 400 × magnification for each experiment performed using NIS elements software. ( C and F ) Graphs showing the quantification of the total VEGFR1 and VEGFR2 present in nuclear and cytoplasmic fractions of the HREC and HAEC after VEGF treatment and control quantified by sandwich ELISA. All graphs represent VEGF time point fold change with respect to control NO-VEGF and the graphs bars represents the average of the a minimum of 3 separate experiments from 3 donors of the HREC and 2 donors of the HAEC. * = p < 0.05 as determined by 1-way ANOVA and multi-comparison post-hoc Tukey’s test was considered significant.

Article Snippet: Total VEGFR levels were quantified using Quantikine sandwich ELISA for Human VEGFR1 (#DVR100C R&D) and PathScan sandwich ELISA for Human VEGFR2 (#7340 Cell signaling) and phosphorylated VEGFRs were quantified using DuoSet IC sandwich ELISA for Human Phospho-VEGFR1 (#DYC4170 R&D) and PathScan sandwich ELISA for Human Phospho-Y1175-VEGFR2 (#7335 Cell signaling) according to the manufacturer’s instructions.

Techniques: Migration, Immunofluorescence, Sandwich ELISA, Blocking Assay, Translocation Assay, Fluorescence, Software

Journal: Scientific Reports

Article Title: Spatial and temporal VEGF receptor intracellular trafficking in microvascular and macrovascular endothelial cells

doi: 10.1038/s41598-021-96964-7

Figure Lengend Snippet: Intravitreal injection of VEGF promotes nuclear localization of VEGFR1 and VEGFR2 in mouse microavascular retinal endothelial cells . Electron microscopy (EM) of VEGFR1 and VEGFR2 immunogold staining in C57BL/6 J mouse retinal endothelial cells. The nuclear and cytoplasmic levels of VEGFRs in retinal endothelial cells from C57BL/6 J mice were evaluated at different time points (2, 6 and 24 h – n = 3/time point) after intravitreal injection of 10 ng VEGF. Negative controls were either: a) untreated eyes or b) eyes that received intravitreal injection of the vehicle (1 ul of the 0.9% saline). ( A ) Double immunogold staining was achieved using two different size of the gold particles to differentiate VEGFR1 (10 nm gold) from VEGFR2 (6 nm). ( B ) Representative EM images from retinal endothelial cells from a control unstimulated eye. ( C ) Negative control showing no specific gold particles in a retinal endothelial cell when the primary antibody was omitted. ( D–E ) Representative EM images from retinal endothelial cells from 2 and 24 h VEGF-stimulated eyes showing expression of VEFR1 (large arrows) and VEGFR2 (small arrows). Scale bar = 200 nm. Graphs show the quantification of the number of gold particles per area (5 µm 2 ) in the nucleus ( F and G ) and cytoplasm ( I and J ) of at least 10 endothelial cells at 11,000 × magnification for each experiment performed using ImageJ software. ( H and K ) The ratio of VEGFR1:VEGFR2 in the nucleus and cytoplasm. Experiments was repeated 2 times for each animal. * = p < 0.05 as determined by 1-way ANOVA and multi-comparison post-hoc Tukey’s test is considered significant.

Article Snippet: Total VEGFR levels were quantified using Quantikine sandwich ELISA for Human VEGFR1 (#DVR100C R&D) and PathScan sandwich ELISA for Human VEGFR2 (#7340 Cell signaling) and phosphorylated VEGFRs were quantified using DuoSet IC sandwich ELISA for Human Phospho-VEGFR1 (#DYC4170 R&D) and PathScan sandwich ELISA for Human Phospho-Y1175-VEGFR2 (#7335 Cell signaling) according to the manufacturer’s instructions.

Techniques: Injection, Electron Microscopy, Staining, Negative Control, Expressing, Software

Journal: Scientific Reports

Article Title: Spatial and temporal VEGF receptor intracellular trafficking in microvascular and macrovascular endothelial cells

doi: 10.1038/s41598-021-96964-7

Figure Lengend Snippet: The nuclear VEGFR1:VEGFR2 ratio is different in micro- and macrovascular endothelial cells and VEGF-stimulated nuclear VEGFRs are minimally phosphorylated . The nuclear levels of VEGFR1 and VEGFR2 plus of phospho-VEGFR1 and phospho-VEGFR2 in HREC and HAEC were evaluated by double immunofluorescence and sandwich ELISA, respectively, at different time points (10 min, 30 min and 2 h) after exposure to 100 ng/ml VEGF and compared to untreated controls (NO-VEGF). ( A ) Representative 3D reconstruction rendering images of the HREC and HAEC after VEGF treatment and control immunostained for full length VEGFR1 (red) + C-terminal VEGFR2 (green) showing simultaneous nuclear localization of both receptors after VEGF treatment. The nuclei were fluorescently stained with the DNA intercalating dye Hoechst (blue) and the blue channel was partially removed from the 3D representations to evidence the simultaneous localization and association of VEGFR1 and VEGFR2 in the area corresponding to the nucleus. The associated points that spatial colocalize green and red are shown in yellow. Scale bars = 5 μm. ( B ) Graphic representation of the nuclear VEGFR1:VEGFR2 ratio of HREC and HAEC following addition of VEGF shown in A after the quantification of the median intensity of fluorescence (MIF) of at least 10 confocal z-stack images at 400 × magnification for each experiment performed using NIS elements software. VEGFR1:VEGFR2 ratio graphs represent VEGF time point fold change with respect to control NO-VEGF and the graphs bars represents the average of the a minimum of 3 separate experiments from HREC and HAEC. Graphs showing the quantification of the phospho-VEGFR1 ( C–D , G–H) and phospho-VEGFR2 ( K–L , O–P ) present in nuclear ( C , G , K and O ) and cytoplasmic ( D, H, L and P ) fractions of the HREC and HAEC after VEGF treatment and control quantified by sandwich ELISA. ( E–F , I–J , M–N , Q–R ) graphic representation of the phospho-VEGFR1 and phospho-VEGFR2 percentage relative to total amount of VEGFR1 or VEGFR2 found in the nucleus and cytoplasm of the HREC and HAEC following addition of VEGF. ELISA data for each VEGF time point was normalized by the control NO-VEGF and bars represent the average of the a minimum of 3 separate experiments from 3 donors of the HREC and 2 donors of the HAEC. * = p < 0.05 as determined by 1-way ANOVA and multi-comparison post-hoc Tukey’s test was considered significant.

Article Snippet: Total VEGFR levels were quantified using Quantikine sandwich ELISA for Human VEGFR1 (#DVR100C R&D) and PathScan sandwich ELISA for Human VEGFR2 (#7340 Cell signaling) and phosphorylated VEGFRs were quantified using DuoSet IC sandwich ELISA for Human Phospho-VEGFR1 (#DYC4170 R&D) and PathScan sandwich ELISA for Human Phospho-Y1175-VEGFR2 (#7335 Cell signaling) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Sandwich ELISA, Staining, Fluorescence, Software, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Spatial and temporal VEGF receptor intracellular trafficking in microvascular and macrovascular endothelial cells

doi: 10.1038/s41598-021-96964-7

Figure Lengend Snippet: VEGFR2 can translocate from plasma membrane and intracellular compartments to the nucleus via early endosomes following VEGF treatment in endothelial cells . Intracellular trafficking of VEGFR2 in HREC and HAEC was evaluated by immunofluorescence at different time points (10 min, 30 min and 2 h) after exposure to 100 ng/ml VEGF and compared to saline controls (NO-VEGF). Triple immunofluorescence was performed to colocalize VEGFR2 with specific organelles. ( A ) 3D reconstruction rendering of the HREC and HAEC immunostained with VEGFR2 (green), trans Golgi (red) and calreticulin (endoplasmic reticulum-ER gray) ( B ) 3D reconstruction rendering of HREC and HAEC immunostained with VEGFR2 (green), LAMP1 (lysosome-red) and EEA1 (early endosome-magenta). The nuclei in ( A ) and ( B ) were fluorescently stained with the DNA intercalating dye Hoechst (blue). The colocalization points between green and red are shown in yellow and between green and magenta shown in gray. The top image of each block shows the 3D rendering of the representative cells of each time point and the same cells are shown in the lower image of each block showing the position of the nuclear translocation of VEGFR2 in only one focal plane in the center and the correlation of this point located in the 3D lateral and lower cross sections. Scale bars = 5 μm. ( C ) Quantification of the number of VEGFR2 colocalized voxels with the organelles relative to the number of cells in each image. The results show the analyzes of at least 10 confocal z-stack images at 400 × magnification for each time point by Imaris software and summarize the data obtained by quantifying the organelles from a minimum of 3 separate experiments from 3 donors of the HREC and 2 donors of the HAEC. The results show that the colocalization of VEGFR2 with trans Golgi and early endosomes increases between 10 and 30 min after VEGF treatment compared to control and correlates with the increase of VEGFR2 in the nucleus in HREC, but not in HAEC. * = p < 0.05 as determined by 1-way ANOVA and multi-comparison post-hoc Tukey’s test is considered significant.

Article Snippet: Total VEGFR levels were quantified using Quantikine sandwich ELISA for Human VEGFR1 (#DVR100C R&D) and PathScan sandwich ELISA for Human VEGFR2 (#7340 Cell signaling) and phosphorylated VEGFRs were quantified using DuoSet IC sandwich ELISA for Human Phospho-VEGFR1 (#DYC4170 R&D) and PathScan sandwich ELISA for Human Phospho-Y1175-VEGFR2 (#7335 Cell signaling) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Blocking Assay, Translocation Assay, Software

Journal: Scientific Reports

Article Title: Spatial and temporal VEGF receptor intracellular trafficking in microvascular and macrovascular endothelial cells

doi: 10.1038/s41598-021-96964-7

Figure Lengend Snippet: Depletion of the biosynthetic pool by Brefeldin A prevents nuclear translocation of VEGFR2 into VEGF-stimulated microvascular endothelial cells . Intracellular trafficking of VEGFR1 and VEGFR2 in HREC and HAEC was evaluated by immunofluorescence at different time points (10 min, 30 min and 2 h) after exposure to 100 ng/ml VEGF and compared to saline controls (NO-VEGF). Triple immunofluorescence was performed to confirm the biosynthetic pool depletion after Brefeldin A treatment and evaluate the nuclear translocation of the VEGFRs by measuring the MIF. ( A–B ) 3D reconstruction rendering of the HREC and HAEC immunostained with VEGFR1 and VEGFR2 (green), respectively, plus trans Golgi (red), calreticulin (endoplasmic reticulum-ER gray) and DNA intercalating dye Hoechst (nuclei-blue). The colocalization points between green and red are shown in yellow and between green and gray shown in cyan. Scale bars = 10 μm. ( C ) Graphs showing the quantification of the median intensity of fluorescence (MIF) volume of at least 10 confocal z-stack images at 400 × magnification for each experiment performed using NIS elements software. MIF graphs represent VEGF time point fold change with respect to control vehicle treated NO-VEGF and the graphs bars represents the average of the a minimum of 3 separate experiments from HREC and HAEC. The results show that the nuclear translocating pool of the VEGFR2 in HREC are derived from the Golgi. * = p < 0.05 as determined by 2-way ANOVA followed by multi-comparison post-hoc Tukey’s test shows intra-group differences with respect to the VEGF treatment and # = p < 0.05 2-way ANOVA followed by multi-comparison post-hoc Tukey’s and t-student tests shows inter-group differences with respect to the Vehicle and Brefeldin A treatment.

Article Snippet: Total VEGFR levels were quantified using Quantikine sandwich ELISA for Human VEGFR1 (#DVR100C R&D) and PathScan sandwich ELISA for Human VEGFR2 (#7340 Cell signaling) and phosphorylated VEGFRs were quantified using DuoSet IC sandwich ELISA for Human Phospho-VEGFR1 (#DYC4170 R&D) and PathScan sandwich ELISA for Human Phospho-Y1175-VEGFR2 (#7335 Cell signaling) according to the manufacturer’s instructions.

Techniques: Translocation Assay, Immunofluorescence, Fluorescence, Software, Derivative Assay

Journal: OncoTargets and therapy

Article Title: Simultaneous Treatment with Soluble Forms of GAS1 and PTEN Reduces Invasiveness and Induces Death of Pancreatic Cancer Cells

doi: 10.2147/OTT.S260671

Figure Lengend Snippet: Characterization of HPAF-II pancreatic cancer cells. ( A ) HPAF-II cells do not express gas1, gfrα1, ret , or gdnf but express both artemin and its receptor gfrα3 . SHSY5Y and U87-MG cells were used as positive controls. ( B ) HPAF-II cells do not express PTEN, as assessed by Western blot analysis; SH-SY5Y cells were used as positive control; β-actin is the loading control.

Article Snippet: ELISA was performed using commercial kits for human Gas1 DuoSet ELISA (R&D Systems, USA; Cat No. DY2636), and for human PTEN (Cell Signaling Technology, USA; Cat No. #7882), according to the manufacturer’s instructions.

Techniques: Western Blot, Positive Control

Journal: OncoTargets and therapy

Article Title: Simultaneous Treatment with Soluble Forms of GAS1 and PTEN Reduces Invasiveness and Induces Death of Pancreatic Cancer Cells

doi: 10.2147/OTT.S260671

Figure Lengend Snippet: Comparison of the effect of soluble forms of GAS1 and PTEN on cell viability. The graphs show the percentage of viable HPAF-II cells, as determined by Trypan blue exclusion at different times after transfection with GAS1, PTEN, or both simultaneously transfected (24, 48, 72 and 96 h). Controls were non-treated HPAF-II cells, cells treated with Lipofectamine or transfected with the Red Fluorescent Protein plasmid (RFP). tGAS1 and PTEN-L were more effective together than when individually transfected. One-way ANOVA followed by Duncan´s test. *p< 0.05, **p<0.01, ***p<0.001; n= 3.

Article Snippet: ELISA was performed using commercial kits for human Gas1 DuoSet ELISA (R&D Systems, USA; Cat No. DY2636), and for human PTEN (Cell Signaling Technology, USA; Cat No. #7882), according to the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation

Journal: OncoTargets and therapy

Article Title: Simultaneous Treatment with Soluble Forms of GAS1 and PTEN Reduces Invasiveness and Induces Death of Pancreatic Cancer Cells

doi: 10.2147/OTT.S260671

Figure Lengend Snippet: Effects of soluble forms of GAS1 and PTEN on intracellular signaling pathways and apoptosis. ( A ) Effects of the soluble forms of GAS1 and PTEN, and when transfected simultaneously on the activity of AKT (pAKT is the phosphorylated molecule and tAKT is the total); ( B ) effects of the soluble forms of GAS1 and PTEN, and transfected together on the activity of ERK1/2 (pERK1/2 is the phosphorylated molecule and tERK1/2 is the total); left panels show representative experiments and right panels, the statistical analysis of three independent experiments; ( C ) apoptosis demonstrated by the activation of caspase-3, β-Actin is the loading control. One-way ANOVA followed by Tukey’s multiple comparisons test. *p< 0.05, **p<0.01, ***p<0.001; n=3.

Article Snippet: ELISA was performed using commercial kits for human Gas1 DuoSet ELISA (R&D Systems, USA; Cat No. DY2636), and for human PTEN (Cell Signaling Technology, USA; Cat No. #7882), according to the manufacturer’s instructions.

Techniques: Transfection, Activity Assay, Activation Assay

Journal: OncoTargets and therapy

Article Title: Simultaneous Treatment with Soluble Forms of GAS1 and PTEN Reduces Invasiveness and Induces Death of Pancreatic Cancer Cells

doi: 10.2147/OTT.S260671

Figure Lengend Snippet: Effect of soluble forms of GAS1 and PTEN, and when simultaneously applied on pancreatic cancer cell invasiveness. ( A ) Levels of the soluble forms of GAS1 and PTEN in conditioned media from HPAF-II producer cells (left transfected with tGAS1; middle with PTEN-L; right with tGAS1 and PTEN-L); ( B ) number of DRG viable cells in the presence of the different media; ( C ) Immunofluorescence of DRG cultures against β-III Tubulin (left panel), nuclei revealed by DAPI (middle panel) and merge of the two channels (right panel). ( D ) Percentage of HPAF-II cancer cells that crossed the membrane towards DRG cells incubated in the presence of the different media; right panel shows a representative experiment. One-way ANOVA followed by Tukey’s multiple comparisons test. **p<0.01, ***p<0.001; n=3.

Article Snippet: ELISA was performed using commercial kits for human Gas1 DuoSet ELISA (R&D Systems, USA; Cat No. DY2636), and for human PTEN (Cell Signaling Technology, USA; Cat No. #7882), according to the manufacturer’s instructions.

Techniques: Transfection, Immunofluorescence, Incubation

Journal: OncoTargets and therapy

Article Title: Simultaneous Treatment with Soluble Forms of GAS1 and PTEN Reduces Invasiveness and Induces Death of Pancreatic Cancer Cells

doi: 10.2147/OTT.S260671

Figure Lengend Snippet: Effects of tGAS1-PTEN-L on tumor cells intracellular pathways and apoptosis. ( A and B ) Levels of Gas1 and PTEN in tumors; ( C ) activity of AKT in tumors, left representative experiment, right statistical analysis; ( D ) activity of ERK1/2 in tumors, left representative experiment, right statistical analysis; ( E ) apoptosis of tumor cells as shown by caspase-3 activity. One-way ANOVA followed by Tukey’s multiple comparisons test. **p<0.01, ***p<0.001; n=3–4 ( A – D ).

Article Snippet: ELISA was performed using commercial kits for human Gas1 DuoSet ELISA (R&D Systems, USA; Cat No. DY2636), and for human PTEN (Cell Signaling Technology, USA; Cat No. #7882), according to the manufacturer’s instructions.

Techniques: Activity Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Chrysin serves as a novel inhibitor of DGK α /FAK interaction to suppress the malignancy of esophageal squamous cell carcinoma (ESCC)

doi: 10.1016/j.apsb.2020.07.011

Figure Lengend Snippet: Chrysin inhibits the expression of downstream malignant effectors via DGK α /FAK complex. (A)–(L) The indicated control or DGK α shRNA or 1 μmol/L PF562271-incubated ESCC cells were treated with 50 μmol/L chrysin. The intratumoral expression of C-MYC (A), cyclin D1 (B), survivin (C), SOX2 (D), NANOG (E), OCT4 (F), BMI1 (G), PKM2 (H), HKII (I), LDHA (J), GLUT1 (K), and the secretion of MMP9 in supernatant (L) were evaluated using quantitative ELISA assay. n.s, no significant difference. Error bars, mean ± SD of five independent experiments.

Article Snippet: For measurement of the expression of intracellular biomarkers in the ESCC cells with the indicated treatments, levels of MYC proto-oncogene protein (C-MYC), G1/S-specific cyclin-D1 (cyclin D1), baculoviral IAP repeat-containing protein 5 (survivin), SRY-box transcription factor 2 (SOX2), nanog homeobox (NANOG), POU class 5 homeobox (OCT4), BMI1 proto-oncogene (BMI1), pyruvate kinase M2 (PKM2), hexokinase 2 (HKII), glucose transporter 1 (GLUT1) or lactate dehydrogenase A (LDHA) in the cell lysates were detected using the human C-MYC ELISA kit (Raybiotech; Cat# ELH-CMYC-1), cyclin D1 ELISA kit (Raybiotech; Cat# ELH-CYCD-1), human survivin ELISA kit (Cell Signaling Technology; Cat# 7169C), human SOX2 ELISA kit (Cell Signaling Technology; Cat# 7277C), human NANOG ELISA kit (Raybiotech; Cat# ELH-NANOG-1), human OCT4 ELISA kit (Raybiotech; Cat# ELH-OCT4-1), human BMI1 ELISA kit (Cell Signaling Technology; Cat# 18157C), human PKM2 ELISA kit (Cloud-Clone; Cat# SEA588HU, Wuhan, China), human HKII ELISA kit (Cloud-Clone; Cat# SED352HU), human GLUT1 ELISA kit (Cloud-Clone; Cat# SEB185HU), or human LDHA ELISA kit (Cloud-Clone; Cat# SEB370HU) according to the manufacturer's instructions.

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