human rh vegf  (R&D Systems)

Journal: Biomedicines

Article Title: Angiopoietin-2 and the Vascular Endothelial Growth Factor Promote Migration and Invasion in Hepatocellular Carcinoma- and Intrahepatic Cholangiocarcinoma-Derived Spheroids

doi: 10.3390/biomedicines12010087

Figure Lengend Snippet: The 3D viability assay. This assay was conducted for HepG2 ( A ), Hep3B ( B ), HuCC-T1 ( C ), and EGI-1 ( D ), which were stimulated with concentrations of 0, 50, 100, 200, 400, and 800 ng/mL of rh-ANG-2 and rh-VEGF, either alone or in combination, for a duration of 48 h. The viability within the 3D cell culture was assessed by quantifying ATP and is indicative of metabolically active cells. ATP levels were represented in histograms as luminescence signals (RLUs). All cell lines exhibited comparable luminescence levels, with the exception of HuCC-T1, which demonstrated lower levels (HepG2: 3.6 × 10 6 ; Hep3B: 3.8 × 10 6 ; HuCC-T1: 9.6 × 10 5 ; EGI-1: 2.4 × 10 6 luminescence units). The experiments were performed in triplicate. Statistical significance between groups was determined using a one-way ANOVA. For all comparisons within different groups, no significant differences were present between untreated (0 ng/mL) and treated spheroids ( p > 0.05).

Article Snippet: Upon formation, spheroids were stimulated immediately (time 0) with recombinant human ANG-2 (rh-ANG-2) (623-AN, R&D Systems, Minneapolis, MN, USA) and/or recombinant human VEGF (rh-VEGF) (293-VE, R&D Systems) proteins at increasing doses (0, 50, 100, 200, 400, 800 ng/mL).

Techniques: Viability Assay, Cell Culture, Metabolic Labelling

Journal: Biomedicines

Article Title: Angiopoietin-2 and the Vascular Endothelial Growth Factor Promote Migration and Invasion in Hepatocellular Carcinoma- and Intrahepatic Cholangiocarcinoma-Derived Spheroids

doi: 10.3390/biomedicines12010087

Figure Lengend Snippet: Migration assay of HCC and CCA spheroids. Migration assays were performed on spheroids from HepG2 ( A , B ), Hep3B ( C , D ), HuCC-T1 ( E , F ), and EGI-1 ( G , H ) cell lines. These were either untreated (Ctrl) or treated with 200 ng/mL of rh-ANG-2, 200 ng/mL of rh-VEGF, or a combination of 100 ng/mL each of rh-ANG-2 and rh-VEGF. Measurements were taken at 3, 24, and 48 h post-treatment. Due to size reduction and cropping, images of EGI-1 spheroids are larger than the frame. Spheroid areas were quantified using Fiji ImageJ to assess migration capability (HepG2 ( B ), Hep3B ( D ), HuCC-T1 ( F ), EGI-1 ( H )). Imaging was performed at 10× magnification. Comparative analyses were conducted to assess differences between the control and treated spheroids at the same time points. We also compared different areas of the spheroids at 24 and 48 h, in contrast to the changes observed at 3 h within the same treatment group. The assays were replicated a minimum of three times. Statistical differences were evaluated using a two-way ANOVA for paired data, with subsequent post hoc testing for multiple comparisons (* p < 0.05, ** p < 0.01).

Article Snippet: Upon formation, spheroids were stimulated immediately (time 0) with recombinant human ANG-2 (rh-ANG-2) (623-AN, R&D Systems, Minneapolis, MN, USA) and/or recombinant human VEGF (rh-VEGF) (293-VE, R&D Systems) proteins at increasing doses (0, 50, 100, 200, 400, 800 ng/mL).

Techniques: Migration, Imaging, Control

Journal: Biomedicines

Article Title: Angiopoietin-2 and the Vascular Endothelial Growth Factor Promote Migration and Invasion in Hepatocellular Carcinoma- and Intrahepatic Cholangiocarcinoma-Derived Spheroids

doi: 10.3390/biomedicines12010087

Figure Lengend Snippet: Migration assay and Western blot of ANG-2 and VEGFA expression following treatment with Trebananib and Bevacizumab. HepG2 ( A ) and HuCC-T1 ( C ) spheroids were left without proangiogenic stimulation (Ctrl and Ctrl plus Trebananib (T)) or treated with 200 ng/mL of rh-ANG-2 either alone or in combination with Trebananib (T). Measurements were taken at intervals of 3, 24, and 48 h post-treatment. The spheroid area was quantified using Fiji ImageJ, serving as a proxy for migration capability (HepG2 ( B ), HuCC-T1 ( D )). For Hep3B spheroids ( E ), which were without proangiogenic stimulation (Ctrl and Ctrl plus Bevacizumab ( B )) or treated with 200 ng/mL of rh-VEGF alone or with Bevacizumab ( B ); measurements were taken at the same time intervals. The spheroid area, indicative of migration capability, was quantified using Fiji ImageJ (Hep3B ( F )). We made comparisons between the spheroids that were treated with rh-ANG-2 or rh-VEGF alone and other treatment groups, evaluating them at the same time intervals. All images were captured at a 10× magnification. Due to the reduction and cropping of images, Hep3B spheroids treated solely with rh-VEGF at 48 h surpass the image boundaries. Protein expression was analyzed in HepG2 ( G ) and HuCC-T1 ( H ) spheroids treated with rh-ANG-2 alone or with Trebananib (T) and in Hep3B ( I ) spheroids treated with rh-VEGF alone or with Bevacizumab ( B ) at 3, 24, and 48 h post-stimulation. We quantified the protein bands using ImageLab 6.1 software. The results from the densitometry are reported as optical density, which we have normalized to β-actin to ensure accurate comparisons. A comparison was made between groups treated with rh-ANG-2 or rh-VEGF alone versus those treated with the addition of Trebananib or Bevacizumab at each time point. These experiments were replicated at least three times to ensure reliability. Statistical differences were determined using a two-way ANOVA, followed by post hoc tests for multiple comparisons. Significance levels are denoted by asterisks: * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Upon formation, spheroids were stimulated immediately (time 0) with recombinant human ANG-2 (rh-ANG-2) (623-AN, R&D Systems, Minneapolis, MN, USA) and/or recombinant human VEGF (rh-VEGF) (293-VE, R&D Systems) proteins at increasing doses (0, 50, 100, 200, 400, 800 ng/mL).

Techniques: Migration, Western Blot, Expressing, Software, Comparison

Journal: Biomedicines

Article Title: Angiopoietin-2 and the Vascular Endothelial Growth Factor Promote Migration and Invasion in Hepatocellular Carcinoma- and Intrahepatic Cholangiocarcinoma-Derived Spheroids

doi: 10.3390/biomedicines12010087

Figure Lengend Snippet: Invasion assay of HCC and CCA spheroids in Matrigel. Spheroids from HepG2 ( A , B ), Hep3B ( C , D ), HuCC-T1 ( E , F ), and EGI-1 ( G , H ) were treated with 200 ng/mL of rh-ANG-2 or rh-VEGF independently and with a combination of 100 ng/mL of rh-ANG-2 and rh-VEGF. Imaging was conducted at 0, 24, and 48 h post-stimulation to monitor the invasion process. The degree of invasiveness of the spheroids was quantified by Fiji ImageJ software. We measured the area they covered after being embedded in Matrigel. Comparative analyses were carried out between the control (Ctrl) and treated spheroids at the same time points. Additionally, within each treatment group, we compared the area of the spheroids at 24 and 48 h with their initial size at time 0. These experiments were performed in triplicate to ensure the robustness of the data. The results are presented as the ratio of the treated spheroids’ area to the baseline, with log2 transformation applied to raw data for normalization. The baseline value was standardized at Y = 1.0. Statistical differences were assessed using a two-way ANOVA for paired data, complemented by post hoc tests for multiple comparisons. Significance is indicated by asterisks (*) for p < 0.05 compared to the baseline within each treatment group and hashes (#) for p < 0.05 compared to the control at the same time points.

Article Snippet: Upon formation, spheroids were stimulated immediately (time 0) with recombinant human ANG-2 (rh-ANG-2) (623-AN, R&D Systems, Minneapolis, MN, USA) and/or recombinant human VEGF (rh-VEGF) (293-VE, R&D Systems) proteins at increasing doses (0, 50, 100, 200, 400, 800 ng/mL).

Techniques: Invasion Assay, Imaging, Software, Control, Transformation Assay

Journal: Biomedicines

Article Title: Angiopoietin-2 and the Vascular Endothelial Growth Factor Promote Migration and Invasion in Hepatocellular Carcinoma- and Intrahepatic Cholangiocarcinoma-Derived Spheroids

doi: 10.3390/biomedicines12010087

Figure Lengend Snippet: Western blot for TIE2 and VEGFR1 receptor expression and activation by phosphorylation. We evaluated the expression of TIE2 and P-TIE2 in HepG2 and HuCC-T1 spheroids ( A – D ) and the expression of VEGFR1 and P-VEGFR1 in Hep3B spheroids ( E , F ). Treatments included 200 ng/mL of rh-ANG-2, 200 ng/mL of rh-VEGF, and a combination of 100 ng/mL of rh-ANG-2 and rh-VEGF. Proteins were harvested at 15, 30, and 60 min post-stimulation. Band intensities were quantified using ImageLab 6.1 software, and densitometry data were presented as optical density values normalized to β-actin. Comparative analyses were performed between the treated spheroids and the controls at identical time points. This experiment was replicated a minimum of three times. Statistical differences were assessed using a two-way ANOVA, with subsequent post hoc tests for multiple comparisons. Significance thresholds were established as follows: * p < 0.05; ** p < 0.001; *** p < 0.001.

Article Snippet: Upon formation, spheroids were stimulated immediately (time 0) with recombinant human ANG-2 (rh-ANG-2) (623-AN, R&D Systems, Minneapolis, MN, USA) and/or recombinant human VEGF (rh-VEGF) (293-VE, R&D Systems) proteins at increasing doses (0, 50, 100, 200, 400, 800 ng/mL).

Techniques: Western Blot, Expressing, Activation Assay, Phospho-proteomics, Software

Journal: Biomedicines

Article Title: Angiopoietin-2 and the Vascular Endothelial Growth Factor Promote Migration and Invasion in Hepatocellular Carcinoma- and Intrahepatic Cholangiocarcinoma-Derived Spheroids

doi: 10.3390/biomedicines12010087

Figure Lengend Snippet: Western blot for EMT marker expressions in HCC and CCA cell lines. We evaluated the expression of E-cadherin, N-cadherin, and Vimentin in HepG2, Hep3B, HuCC-T1, and EGI-1 spheroids. Treatments included 200 ng/mL of rh-ANG-2, 200 ng/mL of rh-VEGF, and a combination of 100 ng/mL of rh-ANG-2 and rh-VEGF. Proteins were harvested at 3 and 48 h post-stimulation. Band intensities were quantified using ImageLab 6.1 software, and densitometry data were presented as optical density values normalized to β-actin. Comparative analyses were performed between the treated spheroids and the controls at identical time points, as well as longitudinally within treatment groups from 3 to 48 h. This experiment was replicated a minimum of three times to ensure reproducibility. Statistical differences were assessed using a two-way ANOVA, with subsequent post hoc tests for multiple comparisons. Significance thresholds were established as * p < 0.05; ** p < 0.001; *** p < 0.001. Comprehensive EMT marker data are summarized in .

Article Snippet: Upon formation, spheroids were stimulated immediately (time 0) with recombinant human ANG-2 (rh-ANG-2) (623-AN, R&D Systems, Minneapolis, MN, USA) and/or recombinant human VEGF (rh-VEGF) (293-VE, R&D Systems) proteins at increasing doses (0, 50, 100, 200, 400, 800 ng/mL).

Techniques: Western Blot, Marker, Expressing, Software