Journal: Bioactive Materials

Article Title: Imidazole functionalized photo-crosslinked aliphatic polycarbonate drug-eluting coatings on zinc alloys for osteogenesis, angiogenesis, and bacteriostasis in bone regeneration

doi: 10.1016/j.bioactmat.2024.03.037

Figure Lengend Snippet: Angiogenic activity of the different Zn alloys samples. (A) Migration assay of EA.hy926 incubated with the extract of different samples after wounding. (B) Quantification statistical analysis of migration assay. (C) Tube formation assay of EA.hy926 in different extracts cultured on Matrigel. (D) Quantification statistical analysis of tube formation assay. (E) Quantification statistical analysis of in vivo CAM assay. (F) Representative photographs of in vivo CAM assay. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The supernatants of differentiated MC3T3-E1 culture system from the blank, and bare Zn, Zn-CP, Zn-CP@SIM groups were collected to culture the human endothelial cell line EA.hy926 (ATCC, CRL-2922) until reaching about 90% confluence.

Techniques: Activity Assay, Migration, Incubation, Tube Formation Assay, Cell Culture, In Vivo, Chick Chorioallantoic Membrane Assay

Journal: The Prostate

Article Title: PSMA‐positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state

doi: 10.1002/pros.24237

Figure Lengend Snippet: PSMA was detected in HUVECs cultured with the conditioned medium derived from LNCaP cells. (A) Confocal images of prostate cancer cells (PC3, DU145, LNCaP cells) cultured in condition 1 or condition 2. The scheme of each condition by which each conditioned medium (CM) was prepared is shown on the right. To prepare the CM, 1.5 × 10 5 cells of prostate cancer cells were seeded on 6‐well plastic dishes (condition 1) or collagen I gels (condition 2). Three days later, the media were replaced with fresh media. The cells were then incubated for another 3 days, then the media were collected as CM. Bars: 100 µm. (B, C) Confocal images of HUVECs cultured with the CM derived from LNCaP cells (B) or PC3 and DU145 cells (C). The CM was diluted to half of its concentration with EBM‐2. The resulting solution was added to the HUVECs seeded on the collagen I gels. Seventy‐two hours later, cells were subjected to immunofluorescence staining for PSMA. Bars: 100 µm. (D) Western blots of HUVEC lysates cultured with the CM derived from prostate cancer cells in condition 2 for 72 h. The CM was diluted to half of its concentration with EBM‐2. The resulting solution was added to the HUVECs seeded on the collagen I gels. The lysates from LNCaP cells were used as a positive control of PSMA expression. (E) The mRNA expression of PSMA in HUVECs cultured with the CM derived from LNCaP cells in condition 2 for 72 h. The CM was diluted to half of its concentration with EBM‐2. The resulting solution was added to the HUVECs seeded on the collagen I gels. Data are mean ± SEM from three independent experiments. ** p < .01. HUVEC, human umbilical vascular endothelial cell; mRNA, messenger RNA; PSMA, prostate‐specific membrane antigen [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Human umbilical vascular endothelial cells (HUVECs) were purchased from Lonza.

Techniques: Cell Culture, Derivative Assay, Incubation, Concentration Assay, Immunofluorescence, Staining, Western Blot, Positive Control, Expressing

Journal: The Prostate

Article Title: PSMA‐positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state

doi: 10.1002/pros.24237

Figure Lengend Snippet: Fractionation of the CM derived from LNCaP cells. (A) Confocal images of HUVECs cultured with each fraction of CM derived from LNCaP cells. Each fraction was diluted to half of its concentration with EBM‐2 or suspended with EBM‐2, and added to the HUVECs seeded on the collagen I gels. Seventy‐two hours later, cells were subjected to immunofluorescence staining for PSMA. The PSMA‐positive HUVECs were shown by arrowheads. Bars: 100 µm. The representative images from three independent experiments were shown. (B) Confocal images of HUVECs cultured with 10,000 g pellet fractions of CM derived from LNCaP cells. The 10,000 g pellet fraction was labeled with a membrane marker dye, before the suspension in EBM‐2 medium. The labeled 10,000 g pellet was cultured with HUVECs for 6 h, and cells were subjected to immunofluorescence staining for PSMA. Bars: 10 µm (left) and 2 µm (right; magnified images of squares in left images). The representative images from three independent experiments were shown. (C) Western blots of 10,000 g pellet fraction of CM derived from LNCaP cells. The lysates from HUVECs and LNCaP cells were used as negative and positive controls of PSMA expression, respectively. The representative blot data from three independent experiments were shown. (D) Confocal images of HUVECs cultured with the CM derived from PC3 cells that stably express Myc‐PSMA or PSMA‐myc. The CM was diluted in half with EBM‐2, and added to the HUVECs seeded on the collagen I gels. Seventy‐two hours later, cells were subjected to immunofluorescence staining for PSMA. Bars: 100 µm. The representative images from three independent experiments were shown. CM, conditioned medium; HUVEC, human umbilical vascular endothelial cell; mRNA, messenger RNA; PSMA, prostate‐specific membrane antigen [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Human umbilical vascular endothelial cells (HUVECs) were purchased from Lonza.

Techniques: Fractionation, Derivative Assay, Cell Culture, Concentration Assay, Immunofluorescence, Staining, Labeling, Marker, Western Blot, Expressing, Stable Transfection

Journal: The Prostate

Article Title: PSMA‐positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state

doi: 10.1002/pros.24237

Figure Lengend Snippet: The tube formation assay of HUVECs cultured with 10,000 g pellet fraction of CM derived from prostate cancer cells. (A) Representative images of tube formation. HUVECs seeded on collagen I gel were treated with the 10,000 g pellet fraction of CM derived from LNCaP cells for 6 h, and packed on collagen I followed by VEGF‐A stimulation for 66 h. HUVECs were stained with Calcein‐AM before acquisition of images. To examine the PSMA dependency, the CM was prepared from LNCaP cells depleted of PSMA. Bars: 100 µm. (B) The quantitation of (A). Total tube lengths from three independent experiments were measured and normalized to those of cells cultured with normal EBM‐2. Data are the means ± SEM . * p < .05; n.s., not significant. (C) Western blots of LNCaP cell lysates, 72 h posttransfection with the indicated siRNAs. (D) Confocal images of PC3 cells stably expressing Myc‐PSMA or PSMA‐myc. Bars: 100 µm. (E) Representative images of tube formation. HUVECs seeded on collagen I gel were treated with the 10,000 g pellet fraction of CM derived from PC3 cells for 6 h, and packed in collagen I followed by VEGF‐A stimulation for 66 h. HUVECs were stained with Calcein‐AM before acquisition of images. Bars: 100 µm. (F) The quantitation of (E). Total tube lengths from three independent experiments were measured and normalized to those of PC3 (parental). Data are the means ± SEM . * p < .05; ** p < .01. CM, conditioned medium; HUVEC, human umbilical vascular endothelial cell; mRNA, messenger RNA; PSMA, prostate‐specific membrane antigen [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Human umbilical vascular endothelial cells (HUVECs) were purchased from Lonza.

Techniques: Tube Formation Assay, Cell Culture, Derivative Assay, Staining, Quantitation Assay, Western Blot, Stable Transfection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Activation of Epidermal Growth Factor Receptor/p38/Hypoxia-inducible Factor-1α Is Pivotal for Angiogenesis and Tumorigenesis of Malignantly Transformed Cells Induced by Hexavalent Chromium *

doi: 10.1074/jbc.M116.715797

Figure Lengend Snippet: Knockdown of EGFR inhibits angiogenesis in Cr(VI)-transformed cells. A, expression of p-EGFR was examined using immunoblotting to confirm stable knockdown of EGFR (shEGFR) in Cr(VI)-transformed cells (BEAS-2B-Cr). B, representative images of tube formation in HUVECs (left panel) and quantitation of tube branches (right panel) induced by conditioned medium from BEAS-2B-Cr cells with (shEGFR) or without (Scramble) EGFR knockdown. *, p < 0.05, compared with scramble cells. C, chick embryos were exposed to conditioned medium from BEAS-2B-Cr cells with (shEGFR) or without (Scramble) EGFR knockdown for 72 h. Top panel, CAM tissue directly beneath each Matrigel graft was photographed. The image represents at least three chick embryos. Bottom panel, quantitation of vascular branches in CAM. The results were expressed as means ± S.D. *, p < 0.05, compared with scramble cells. D, in vivo Matrigel plug assay. Top panel, representative pictures of Matrigel plugs from BEAS-2B-Cr cells with (shEGFR) or without (Scramble) EGFR knockdown at day 14 after inoculation into mice. Bottom panel, hemoglobin level of Matrigel plugs. The data are means ± S.D. from replicate experiments (n = 3). *, p < 0.05, compared with scramble cells.

Article Snippet: Antibodies against EGFR, p -EGFR, MMP-1, p38, p -p38, p42/44, p -p42/44, and PH2/EGL-9 and p38 inhibitor SB203580 were from Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Transformation Assay, Expressing, Western Blot, Quantitation Assay, In Vivo, Matrigel Assay

Journal: The Journal of Biological Chemistry

Article Title: Activation of Epidermal Growth Factor Receptor/p38/Hypoxia-inducible Factor-1α Is Pivotal for Angiogenesis and Tumorigenesis of Malignantly Transformed Cells Induced by Hexavalent Chromium *

doi: 10.1074/jbc.M116.715797

Figure Lengend Snippet: Knockdown of EGFR reduces MMP-1 and VEGF expression in Cr(VI)-transformed cells. A, Cr(VI)-transformed cells (BEAS-2B-Cr) with (shEGFR) or without (Scramble) stable knockdown of EGFR were examined for MMP-1 and VEGF expression using immunoblotting analysis. B, VEGF level was measured in BEAS-2B-Cr cells with (shEGFR) or without (Scramble) EGFR knockdown using ELISA. *, p < 0.05, compared with scramble cells. C, Cr(VI)-transformed cells with (shEGFR) or without (Scramble) stable EGFR knockdown were grown in chamber slides. Expression of MMP-1 (green) and VEGF (red) was examined using immunofluorescence staining. The images were captured using fluorescence microscope. Expression of DAPI (blue) was used as nuclear control. D, quantitative PCR analysis. mRNA levels of angiogenin, IL-6, MMP-1, and GM-CSF were examined in BEAS-2B-Cr cells with (shEGFR) or without (Scramble) stable EGFR knockdown. *, p < 0.05, compared with scramble cells.

Article Snippet: Antibodies against EGFR, p -EGFR, MMP-1, p38, p -p38, p42/44, p -p42/44, and PH2/EGL-9 and p38 inhibitor SB203580 were from Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Expressing, Transformation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Fluorescence, Microscopy, Control, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Activation of Epidermal Growth Factor Receptor/p38/Hypoxia-inducible Factor-1α Is Pivotal for Angiogenesis and Tumorigenesis of Malignantly Transformed Cells Induced by Hexavalent Chromium *

doi: 10.1074/jbc.M116.715797

Figure Lengend Snippet: Inhibition of EGFR down-regulates HIF-1α and p38 in Cr(VI)-transformed cells. A, Cr(VI)-transformed cells (BEAS-2B-Cr) and passage-matched normal ones (BEAS-2B) were collected for immunoblotting analysis. B, immunofluorescence staining analysis in normal lung tissue from a health patient and in lung tumor tissue and its normal adjacency from a worker exposed to Cr(VI) for 19 years as described under “Experimental Procedures.” C, binding of HIF-1α to hypoxia response element region of VEGF promoter was examined using ChIP assay. Data are means ± S.D. (n = 3). * and #, p < 0.05, compared with BEAS-2B cells and BEAS-2B-Cr scramble cells. D, BEAS-2B-Cr cells with (shEGFR) and without (Scramble) (left panel) and tumor tissues injected with stable knockdown EGFR (shEGFR) and scramble (right panel) were subjected to immunoblotting analysis. E, BEAS-2B-Cr cells were treated with SB203580 or PX478 for 24 h. Whole cell lysates were harvested for immunoblotting analysis. F, tube formation assay. Conditioned medium were collected from BEAS-2B-Cr cells treated with SB203580 or PX478 for 24 h or without treatment. HUVECs were plated onto Matrigel-coated plates and incubated with those conditioned medium. Top panel, representative images from each treatment group. Bottom panel, quantitative results of tube formation are shown as mean ± S.D. (n = 3). *, p < 0.05, compared with BEAS-2B-Cr cells without treatment. G, BEAS-2B-Cr cells were transient transfected with HIF-1α shRNA for 48 h. Whole cell lysates were harvested for immunoblotting analysis. H, Cr(VI)-transformed cells were transfected with EGFR expression plasmid and/or EGFR shRNA for 48 h in various combinations. The cells were collected, and whole cell lysates were subjected for immunoblotting analysis.

Article Snippet: Antibodies against EGFR, p -EGFR, MMP-1, p38, p -p38, p42/44, p -p42/44, and PH2/EGL-9 and p38 inhibitor SB203580 were from Cell Signaling Technology (Danvers, MA).

Techniques: Inhibition, Transformation Assay, Western Blot, Immunofluorescence, Staining, Binding Assay, Injection, Knockdown, Tube Formation Assay, Incubation, Transfection, shRNA, Expressing, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Activation of Epidermal Growth Factor Receptor/p38/Hypoxia-inducible Factor-1α Is Pivotal for Angiogenesis and Tumorigenesis of Malignantly Transformed Cells Induced by Hexavalent Chromium *

doi: 10.1074/jbc.M116.715797

Figure Lengend Snippet: Schematic mechanism of angiogenesis and tumorigenesis of Cr(VI)-transformed cells. Our previous study has demonstrated that chronic exposure of cells to Cr(VI) causes malignant cell transformation and EGFR is constitutively activated in Cr(VI)-transformed cells. The present study has found that constitutive activation of EGFR causes activations of p38 and HIF-1α, leading to angiogenesis and tumorigenesis of Cr(VI)-transformed cells.

Article Snippet: Antibodies against EGFR, p -EGFR, MMP-1, p38, p -p38, p42/44, p -p42/44, and PH2/EGL-9 and p38 inhibitor SB203580 were from Cell Signaling Technology (Danvers, MA).

Techniques: Transformation Assay, Activation Assay

Journal: Oncotarget

Article Title: Treatment of diabetes mellitus-induced erectile dysfunction using endothelial progenitor cells genetically modified with human telomerase reverse transcriptase

doi: 10.18632/oncotarget.9909

Figure Lengend Snippet: (A) Rat BM-MNCs from bone marrow after 7 days in culture (100×). (B) Rat BM-MNCs after 14 days of culture (100×). (C) CD31, CD34, CD133 and VEGFR-2 expression was detected using immunofluorescence (200×). (D) EPCs differentiation potential into adipocytes was demonstrated using oil red-O staining (100×). (E) EPCs differentiation potential into smooth muscle cells was demonstrated using immunofluorescence staining (200×). (F) The expression of eNOS in EPCs was identified using Western blotting analyses. Human umbilical vein endothelial cells was used as positive control. Cavernous smooth muscle cells was used as negative control. (G) Tube formation assay was examined microscopically (100×).

Article Snippet: Membranes were blocked with 5% BSA for 2 hours and incubated with the following primary antibodies: TERT (1:1000, Affinity), VEGF (1:1000, ProteinTech), bFGF (1:1000, Affinity), IGF-1 (1:1000, ProteinTech), TGF-β1 (1:1000, Abcam, Cambridge, UK), Smad2/3 (1:1000, Abcam), phospho-Smad2/3 (p-Smad2/3, 1:1000, Abcam), Bcl-2 (1:1000, Affinity), Bax (1:1000, ProteinTech), eNOS (1:1000, Abcam), p-eNOS (1:1000, CST, Danvers, USA), and nNOS (1:1000, Abcam).

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Positive Control, Negative Control, Tube Formation Assay

Journal: Oncotarget

Article Title: Treatment of diabetes mellitus-induced erectile dysfunction using endothelial progenitor cells genetically modified with human telomerase reverse transcriptase

doi: 10.18632/oncotarget.9909

Figure Lengend Snippet: (A) Western blotting was used to detect the expression of eNOS and nNOS. (B) Bar graphs show the relative expression of eNOS and nNOS normalized to β-actin levels. (C) Cavernous NO levels were determined. (D) Cavernous cGMP levels were determined. *P<0.05 vs. normal control group, # P<0.05 vs. DMED group, & P<0.05 vs. EPCs group and EPCs-control group.

Article Snippet: Membranes were blocked with 5% BSA for 2 hours and incubated with the following primary antibodies: TERT (1:1000, Affinity), VEGF (1:1000, ProteinTech), bFGF (1:1000, Affinity), IGF-1 (1:1000, ProteinTech), TGF-β1 (1:1000, Abcam, Cambridge, UK), Smad2/3 (1:1000, Abcam), phospho-Smad2/3 (p-Smad2/3, 1:1000, Abcam), Bcl-2 (1:1000, Affinity), Bax (1:1000, ProteinTech), eNOS (1:1000, Abcam), p-eNOS (1:1000, CST, Danvers, USA), and nNOS (1:1000, Abcam).

Techniques: Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: Dehydrocostuslactone Suppresses Angiogenesis In Vitro and In Vivo through Inhibition of Akt/GSK-3β and mTOR Signaling Pathways

doi: 10.1371/journal.pone.0031195

Figure Lengend Snippet: A, HUVECs were treated with or without DHC (0.3–10 µM) in EGM-2 medium. After 72 h of incubation, cells were stained with crystal violet and determined the inhibition of cell proliferation by the absorbance at 550 nm. B, DNA synthesis was assessed by BrdU incorporation assay. C, representative photographs of capillary-like structures formation of CTL and DHC-treated HUVECs on matrigel under microscope (magnification is X100). D, Quantification of the total tube length of capillary-like structures by image analysis software. Data represent the mean ± SEM from three independent experiments. ** P <0.01 and *** P <0.001 versus control. E, effect of DHC on cell migration using a transwell assay. F, the graph shows quantitative analysis of the migrated cell numbers in tranwell assay. Data represent the mean ± SEM from three independent experiments. ## P <0.01 versus basal.

Article Snippet: Endothelial cell basal medium (EBM) and endothelial growth factors (EGM-2) were purchased from Clonetics (BioWhittaker, Walkersville, MD).

Techniques: Incubation, Staining, Inhibition, DNA Synthesis, BrdU Incorporation Assay, Microscopy, Software, Migration, Transwell Assay

Journal: PLoS ONE

Article Title: Dehydrocostuslactone Suppresses Angiogenesis In Vitro and In Vivo through Inhibition of Akt/GSK-3β and mTOR Signaling Pathways

doi: 10.1371/journal.pone.0031195

Figure Lengend Snippet: A, after starvation for 24 hr, HUVECs were pretreated with or without DHC for 60 min and then incubated with EGM-2 at indicated time. Cells were harvested, analyzed by western blot and probe with antibodies. B, cyclin D1 expression was inhibited by DHC in a concentration dependent manner. Actin expression served as a loading control. Data represent from three independent experiments.

Article Snippet: Endothelial cell basal medium (EBM) and endothelial growth factors (EGM-2) were purchased from Clonetics (BioWhittaker, Walkersville, MD).

Techniques: Incubation, Western Blot, Expressing, Concentration Assay

Journal: PLoS ONE

Article Title: Dehydrocostuslactone Suppresses Angiogenesis In Vitro and In Vivo through Inhibition of Akt/GSK-3β and mTOR Signaling Pathways

doi: 10.1371/journal.pone.0031195

Figure Lengend Snippet: A, Western blot analysis of Akt phosphorylation inhibited by DHC with the indicated times and concentrations. B, HUVECs were transfected with either vector or myr-Akt, starved for 24 hr, and then pretreated with DHC for 1 hr followed by incubation in EGM-2 medium for 10 min. Cells were harvested and analyzed protein expression by western blot. C and D, western blot analysis of cyclin D1 expression and GSK-3β phosphorylation in HUVECs transfected with the indicated plasmids and then treated with DHC (3 and 5 µM) and Ly294002 (10 µM) for 6 hr. Data represent from three independent experiments.

Article Snippet: Endothelial cell basal medium (EBM) and endothelial growth factors (EGM-2) were purchased from Clonetics (BioWhittaker, Walkersville, MD).

Techniques: Western Blot, Transfection, Plasmid Preparation, Incubation, Expressing

Journal: PLoS ONE

Article Title: Dehydrocostuslactone Suppresses Angiogenesis In Vitro and In Vivo through Inhibition of Akt/GSK-3β and mTOR Signaling Pathways

doi: 10.1371/journal.pone.0031195

Figure Lengend Snippet: A, crystal violet assay. Treatment of vector group with DHC (3 µM) in EGM-2 medium significantly decreased cell proliferation numbers compared with untreated vector group. Akt overexpression significantly increased cell proliferation in DHC-treated group. B, tube formation assay. Treatment of vector group with DHC (3 µM) abrogated tube formation compared with vector control group. Akt overexpression partial reversed the inhibitory effect. C, crystal violet assay. HUVECs were treated with DHC (3 µM) and/or LiCl (10 mM). NaCl (10 mM) was as an osmolality control. Data represent from three independent experiments.

Article Snippet: Endothelial cell basal medium (EBM) and endothelial growth factors (EGM-2) were purchased from Clonetics (BioWhittaker, Walkersville, MD).

Techniques: Crystal Violet Assay, Plasmid Preparation, Over Expression, Tube Formation Assay

Journal: PLoS ONE

Article Title: Dehydrocostuslactone Suppresses Angiogenesis In Vitro and In Vivo through Inhibition of Akt/GSK-3β and mTOR Signaling Pathways

doi: 10.1371/journal.pone.0031195

Figure Lengend Snippet: A, Western blot analysis of phosphorylation of mTOR, p70S6K, eIF4E and 4EBP in HUVECs treated with DHC for the indicated times and concentrations. B, after transfected with the indicated plasmids, HUVECs were starved for 24 hr and then pretreated with DHC followed by 10 min of EGM-2 incubation. Phosphorylation of mTOR and 4EBP were analyzed by western blot. Data represent from three independent experiments.

Article Snippet: Endothelial cell basal medium (EBM) and endothelial growth factors (EGM-2) were purchased from Clonetics (BioWhittaker, Walkersville, MD).

Techniques: Western Blot, Transfection, Incubation

Journal: The Journal of biological chemistry

Article Title: The YAP-TEAD4 complex promotes tumor lymphangiogenesis by transcriptionally upregulating CCBE1 in colorectal cancer.

doi: 10.1016/j.jbc.2023.103012

Figure Lengend Snippet: Figure 3. CCBE1 mediates the protumor lymphangiogenic function of YAP/TAZ in vitro and in vivo. A, Western blot analysis of CCBE1 protein levels in supernatants from the indicated stable SW837 cells. B, Western blot analysis of pro-VEGFC and mature VEGFC protein levels in the indicated mixed conditioned medium. Conditioned medium from the indicated treated SW837 cells was mixed with conditioned medium from VEGFC-expressing 293T cells (1:1), incubated overnight, and analyzed by Western blotting. Ponceau S staining was used to control for equal loading. C, tube formation and wound healing assays of human lymphatic endothelial cells (HLECs) cultured with conditioned medium from the indicated SW837 cells. The scale bars for the tube formation assay represent 100 μm. The scale bars for the wound healing assay represent 200 μm. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test. D, immunohistochemical analysis of mLyve-1 (lymphatic vessel number) in the indicated HCT116 cell line–derived xenografts in mouse footpads. E, repre- sentative images of mLyve-1(+) lymphatic vessels and CCBE1 protein expression in the same field are shown. Black arrows: mLyve-1(+) lymphatic vessels. Stars: colorectal cancer cells. The scale bars represent 20 μm. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test.

Article Snippet: The following antibodies and reagents were obtained commercially: anti-CCBE1 antibody (Atlas Antibodies, HPA041374, for IHC and WB), anti-VEGFC antibody (Santa Cruz Biotechnology, sc-374628), anti-YAP antibody (Santa Cruz Biotechnology, sc-101199, for IHC and WB), anti-YAP/TAZ antibody (Cell Signaling Technology, D24E4, for WB), anti-BRD4 antibody (Cell Signaling Technology, E2A7X), anti-FLAG antibody (DYKDDDDK Tag, Cell Signaling Technology, D6W5B), anti-human D2-40 (PDPN) antibody (Dako), anti-mouse Lyve-1 antibody (eBioscience, ALY7), Human VEGFC (Pepro Tech, 100-20C), and JQ1 (Selleckchem, S7110).

Techniques: In Vitro, In Vivo, Western Blot, Expressing, Incubation, Staining, Control, Cell Culture, Tube Formation Assay, Wound Healing Assay, Immunohistochemical staining, Derivative Assay

Journal: The Journal of biological chemistry

Article Title: The YAP-TEAD4 complex promotes tumor lymphangiogenesis by transcriptionally upregulating CCBE1 in colorectal cancer.

doi: 10.1016/j.jbc.2023.103012

Figure Lengend Snippet: Figure 4. JQ1 inhibits lymphangiogenesis by suppressing the expression of CCBE1 in CRC. A, JQ1 decreased the mRNA level of CCBE1 in HCT116 and SW837 cells and primary cancer-associated fibroblasts derived from two different CRC tissues. Cells were treated with JQ1 (1 μM) or DMSO for 24 h, and the mRNA level of CCBE1 was then determined by quantitative. **p < 0.01, ****p < 0.0001 by Student’s t test. B, knockdown of BRD2/3/4 by siRNAs decreased the mRNA level of CCBE1 in HCT116 and SW837 cells. Cells were transfected with the indicated siRNA for 72 h, and the mRNA level of CCBE1 was then determined by quantitative PCR. ****p < 0.0001 by Student’s t test. C, Western blot analysis of CCBE1 protein levels in supernatants from the indicated SW837 cells treated with JQ1 (1 μM) or DMSO for 24 h. D, Western blot analysis of pro-VEGFC and mature VEGFC protein levels in the indicated mixed conditioned medium. Conditioned medium from the indicated treated SW837 cells was mixed with conditioned medium from full-length VEGFC-expressing 293T cells (1:1), incubated overnight and analyzed by Western blotting. E, Western blot analysis of CCBE1 protein levels in supernatants from the indicated treated SW837 cells overexpressing CCBE1 and treated with JQ1 (1 μM) or DMSO for 24 h. F, Western blot analysis of pro-VEGFC and mature VEGFC protein levels in the indicated mixed conditioned medium. Conditioned medium from the indicated treated stable SW837 cells was mixed with conditioned

Article Snippet: The following antibodies and reagents were obtained commercially: anti-CCBE1 antibody (Atlas Antibodies, HPA041374, for IHC and WB), anti-VEGFC antibody (Santa Cruz Biotechnology, sc-374628), anti-YAP antibody (Santa Cruz Biotechnology, sc-101199, for IHC and WB), anti-YAP/TAZ antibody (Cell Signaling Technology, D24E4, for WB), anti-BRD4 antibody (Cell Signaling Technology, E2A7X), anti-FLAG antibody (DYKDDDDK Tag, Cell Signaling Technology, D6W5B), anti-human D2-40 (PDPN) antibody (Dako), anti-mouse Lyve-1 antibody (eBioscience, ALY7), Human VEGFC (Pepro Tech, 100-20C), and JQ1 (Selleckchem, S7110).

Techniques: Expressing, Derivative Assay, Knockdown, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Incubation

Journal: Gene

Article Title: The Mef2c/AdipoR1 axis is responsible for myogenic differentiation and is regulated by resistin in skeletal muscles.

doi: 10.1016/j.gene.2023.147193

Figure Lengend Snippet: Fig. 4. Mef2c binding on the promoter of adiponectin receptor 1. (A) Bioinformatic analysis showed a mef2c binding site of −430 bp on the promoter of adipoR1, (B) C2C12 cells was transfected with mef2c plasmid (pcDNA3.1-mef2c) and adiponectin and adiponectin R1 expression were identified by western blot, as well as the adiponectin R2 expression level. (C) The promoter sequence of adiponectin R1 was amplified to generate the luciferase report vector including a wild-type vector (pGL-full length AdipoR1p) and a vector carrying mutations in putative mef2c binding sites(pGL-mut-AdipoR1p). (D) pGL-full length AdipoR1p and pGL-mut- AdipoR1p were co-transfected with mef2c plasmid, respectively, and the luciferase activity were detected. (E) Chromatin immunoprecipitation assays revealed that mef2c interacts specifically with the binding site on adiponectin R1 promoter. Data are represented as mean ± SD. *p < 0.05, **p < 0.01 and *** p < 0.001. Values with different letters are significantly different (p < 0.50).

Article Snippet: The pellets were resuspended in lysis buffer and the samples were aliquoted into 1 mL/ tube and then incubated with Mef2c antibodies (CST, WWLP, USA) overnight at 4 ◦C and rotated continuously.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Sequencing, Amplification, Luciferase, Activity Assay, Chromatin Immunoprecipitation

Journal: Gene

Article Title: The Mef2c/AdipoR1 axis is responsible for myogenic differentiation and is regulated by resistin in skeletal muscles.

doi: 10.1016/j.gene.2023.147193

Figure Lengend Snippet: Fig. 5. Resistin inhibits myoblast differentiation of C2C12 through an mef2c-AdiopR1 signal pathway. (A) The C2C12 cells were cultured and treated with or without resistin, and phosphorylated mef2c and adiponectin R1 protein level were analyzed by western bolt. (B) C2C12 cell were transfected with mef2c or adipoR1 plasmid, respectively, and treated with or without resistin recombinant protein, then induced to differentiation, MHC immunofluorescent staining was used to identify the differentiation. Data are represented as mean ± SD. *p < 0.05, **p < 0.01 and *** p < 0.001. Values with different letters are significantly different (p < 0.50).

Article Snippet: The pellets were resuspended in lysis buffer and the samples were aliquoted into 1 mL/ tube and then incubated with Mef2c antibodies (CST, WWLP, USA) overnight at 4 ◦C and rotated continuously.

Techniques: Cell Culture, Western Blot, Transfection, Plasmid Preparation, Recombinant, Staining

Journal: Gene

Article Title: The Mef2c/AdipoR1 axis is responsible for myogenic differentiation and is regulated by resistin in skeletal muscles.

doi: 10.1016/j.gene.2023.147193

Figure Lengend Snippet: Fig. 6. The proposed pathway of resistin regulate myogenic differentiation through mef2c-adipoR1. This scheme illustrates the potential mechanisms of resistin regulate myogenic differentiation by mef2c. In normal physiological state, adiponectin promotes myogenic differentiation and maintain muscle tissue of normal physiological function through adiponectin receptor 1 signaling pathway. While, in some cases, such as obesity, diabetes, elevated resistin level effects on muscle cells and decrease the expression of cytokines-mef2c and its phosphorylation level, consequently, the interaction between mef2c and adiponectin receptor 1 promoter decreased, resulting in inhibition of myocyte differentiation and even its function.

Article Snippet: The pellets were resuspended in lysis buffer and the samples were aliquoted into 1 mL/ tube and then incubated with Mef2c antibodies (CST, WWLP, USA) overnight at 4 ◦C and rotated continuously.

Techniques: Expressing, Phospho-proteomics, Inhibition

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 maintains BTIC self-renewal. ( a ) Morphology of self-renewing spheres and immunofluorescence of neuronal and glial lineage markers in differentiated cells. Scale bar=200 μm. ( b ) Immunofluorescence of ING5 in undifferentiated (upper panels) and cells differentiated for 5 days (lower panels). Scale bar=20 μm. ( c , d ) The mRNA levels ( c ) and protein levels ( d ) of ING5 decrease during differentiation in BT 189 cells. n =4. ( e ) Immunoblotting of ING5 protein in the BT 12, and BT 134 lines during differentiation. ( f ) ING5 overexpression increases sphere formation rates and average volumes in BTIC sphere formation assays ( n =3, ** P <0.01, * P <0.05). Scale bar=400 μm. ( g ) ING5 knockdown by siRNA decreases sphere formation rates and sphere sizes of BTICs ( n =4, ** P <0.01, * P <0.05). Scale bars=400 μm. ( h ) (Left) Sphere formation rates for cell lines stably expressing shRNAs against ING5 (shR1 and shR2-ING5) or control non-targeting shRNA (shR-ctr). ( n =4, ** P <0.01, * P <0.05). (Right) Fluorescence of the RFP reporter in stable cell lines superimposed with differential interference contrast (DIC) images. Scale bar=100 μm. ( i ) RT-qPCR analysis of stem cell core transcription factors and stem cell markers after ING5 overexpression. ( j , k ) Western blot analysis of the neural stem cell marker Nestin and neuronal lineage marker Tubb3 in response to ING5 overexpression ( j ) and knockdown ( k ).

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Immunofluorescence, Western Blot, Over Expression, Knockdown, Stable Transfection, Expressing, Control, shRNA, Fluorescence, Quantitative RT-PCR, Marker

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 increases the stem cell pool and inhibits differentiation. ( a ) Flow cytometry analysis of CD133/CD44 positive cells in iPB cell lines (top panels) and CD44 positive cells in shRNA cell lines (bottom panels), gated by isotype control. ( b ) (Left) Mitotic pair analysis of the three division modes: symmetric proliferating (sym-pro), symmetric differentiating (sym-diff) and asymmetric (asym) cell division, in iPB cell lines. Over 150 pairs were counted for each group in one experiment. n =3, * P <0.05. (Right) An example of cell division symmetry based on the distribution of stem cell factor Nestin with the Red arrow indicating symmetric differentiating division and the white arrow asymmetric division. ( c ) Morphological changes of iPB cells before (Day 0) and after (Days 1–3) differentiation induced by 1% FBS. Scale bar=100 μm. ( d ) After differentiation for 5 days, immunofluorescence of Nestin and Tubb3 in shRNA cell lines with an RFP reporter. Scale bar=200 μm. ( e ) Western blot of Nestin and Tubb3 in differentiated shRNA cell lines corresponding to cells shown in d .

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Flow Cytometry, shRNA, Control, Immunofluorescence, Western Blot

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 activates mitogenic pathways to promote self-renewal. ( a ) The sphere formation rates of iPB-ctr and iPB-ING5 overexpressing cells at three successive passages in the absence of EGF and FGF treatment. ( n =3, ** P <0.01, *** P <0.001). ( b ) DIC images of spheres from the tertiary sphere passage in iPB cells. Scale bar=400 μm. ( c ) Sphere formation rate under treatment with protein kinase inhibitors ( n =3, * P <0.05). ( d ) Protein and phosphorylated protein levels of effectors in the PI3K and MEK pathways. Cells were treated with PX-866 at 1 μ M and PD184352 at 2 μ M for 48 h. ( e ) Immunofluorescence of Nestin and Tubb3 in differentiated iPB control (Left panels) or iPB-ING5 cells (Right panels) treated with 1 μ M PX-866 or 2 μ M PD184352. Scale bar=200 μm. ( f ) Flow cytometry analysis of the CD133 positive population in PX-866 (1 μ M ) and PD184352 (2 μ M ) treated BTIC 189 cells, gated by isotype control.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Immunofluorescence, Control, Flow Cytometry

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: The FSH pathway transduces effects of ING5 on stem cell properties. ( a ) Sphere formation assay for cells treated with calcium modulators and FSHR blocking antibody (Anti-FSHR) at the indicated concentrations ( n =3, * P <0.05 and ** P <0.01 compared to iPB-ctr/DMSO; # P <0.05 and ## P <0.01 compared to iPB-ING5/DMSO). ( b ) IPA downstream function analysis indicates the FSH pathway is elevated by ING5. Genes positively correlated with this function were listed with fold changes. ( c ) RT-qPCR of genes related to hormone and steroidogenesis functions. ( n =3, * P <0.05, ** P <0.01) ( d ) The expression levels of FSHB and FSHR genes in BT 189 cells before and after differentiation for 1–5 days. ( e ) Immunostaining for ING5 and FSHR in iPB cells. Scale bar=100 μm. ( f ) Flow cytometry analysis of CD133 positive cells in BT 189 cells treated with FSHR neutralizing antibody or IgG control, gated by isotype control. ( g ) Immunofluorescence of Nestin and Tubb3 shows inhibition of the FSH pathway induces neuronal differentiation. Scale bar=200 μm. ( h ) FSH recombinant protein treatment at indicated concentrations increases sphere formation rates in shRNA cell lines ( n =3, * P <0.05 and ** P <0.01 compared to untreated shR-ctr; # P <0.05 compared to untreated shR-ING cells). ( i ) FSH recombinant protein treatment induces sphere-forming abilities in iPB-ctr cells but not in ING5 overexpressing cells ( n =3, ** P <0.01). ( j ) Sphere formation rates for cells treated with Anti-FSHR or BAPTA alone, and the combination of both ( n =3, * P <0.05). ( k ) FSH treatment at 5 ng/ml for 3 days induces the expression of OCT4 and Nestin in BT 189 cells.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Tube Formation Assay, Blocking Assay, Quantitative RT-PCR, Expressing, Immunostaining, Flow Cytometry, Control, Immunofluorescence, Inhibition, Recombinant, shRNA

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: PHD motif is required for the function of ING5 in BTICs and ING5 levels negatively correlate with survival of GBM. ( a ) Sphere formation assays in iPB cell lines overexpressing wild-type (ING5-FLAG) and PHD-deleted ING5 (ΔPHD) ( n =3, ** P <0.01). ( b ) Western blot shows the protein levels of endogeneous ING5, wildtype ING5 with a FLAG tag and PHD-deleted ING5 (black arrows) in three iPB cell lines. ( c ) ChIP analysis of ING5 binding to promoters of target genes presented as fold enrichment relative to IgG controls. The endogenous ING5 in BT 189 cells, overexpressed ING5 with a Flag tag in iPB-ING5 cells and overexpressed PHD-deleted ING5 protein with a Flag tag were immunoprecipitated by the ING5 antibody and Flag antibody respectively. The upper panels are the schematic representation of the location of the primer sets and promoter regions enriched for ING5 binding were shown in red. ( d ) Kaplan–Meier survival analysis of TCGA GBM patients with high and low levels of ING5 expression (stratified by mean value, n =114). ( e , f ) ING5 expression levels negatively correlate with survival of the Proneural subtype ( n =24) and the Classical subtype ( n =30) of GBM patients. ( g ) The relationship of ING5 levels to survival in the SOX2-low group of patients (ING5, SOX2 stratified by median values, n =61). ( h ) Model for how ING5 functions in the maintenance of BTIC self-renewal. In the absence of growth factors, ING5 induces FSH and calcium signaling by promoting transcription of the FSH receptor and ligand genes, and various plasma membrane calcium channel genes. The FSH and calcium signaling pathways further activate PI3K/AKT and MEK/ERK signaling to induce stem cell features and the expression of stemness factors OCT4, OLIG2 and Nestin. Gene activation by ING5 is dependent on its PHD motif to target ING5-associated histone acetyltransferase complexes to the promoters.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Western Blot, FLAG-tag, Binding Assay, Immunoprecipitation, Expressing, Clinical Proteomics, Membrane, Protein-Protein interactions, Activation Assay

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 1. Upregulation of ALDH1A3 in oxGBM cells is associated with the increased expression and release of pro-angiogenic factors PAI-1 and IL-8. GBM cell lines were transduced with ALDH1A3 for overexpression (oxGBM) or with empty vector (evGBM). (A) Confirmation of up-regulation of ALDH1A3 mRNA level in oxGBM cells by RT2-PCR. (B) Confirmation of up-regulation of ALDH1A3 protein expression by Western blot. wt, wild-type cells. The uncropped blots are shown in Figure S7; (C) Angiogenesis array. The blots showed duplicated dots for 55 angiogenesis-related proteins in the media of oxU373 or evU373. 10 of 55 proteins were upregulated more than 2-fold in ox group compared to ev group (indicated by rectangle). They are: (1) Ang-1, (2) artemin, (3) TF, (4) ET-1, (5) GM-CSF, (6) IL-8, (7) PDGF-AA, (8) PAI-1, (9) PEDF, and (10) uPA. (D) Semi-quantification of the dots representing PAI-1 and IL-8. (E) Immunofluorescence staining of GBM cells. U373 (left panel) and LN229 (right panel). Co-localization of ALDH1A3 with PAI-1 and IL-8 was observed in oxGBM cells, whereas no immunoreactivity of PAI-1 and IL-8 was detected in evGBM cells. (F) mRNA expression of PAI-1 and IL-8 in transduced U373 cells and the effect of inhibitors. Tiplaxtinin (Tip, 30 µM) and reparixin (Rep, 1 µM) are the specific inhibitors of PAI-1 and IL-8 receptors CXCR1/2, respectively. (G) Detection of PAI-1 and potential signaling proteins by Western blot. IOD: optical density. The uncropped blots are shown in Figure S8. **, p < 0.01; ***, p < 0.001, compared with ev. ##, p < 0.01; ###, p < 0.001, compared with ox.

Article Snippet: To evaluate the potential angiogenic factors released from oxGBMs, an array was carried out using a human angiogenesis array kit (cat# ARY007; R&D Systems, Wiesbaden, Germany) according to the manufacturer’s protocol.

Techniques: Expressing, Transduction, Over Expression, Plasmid Preparation, Western Blot, Staining

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 2. Overexpression of ALDH1A3 in GBM cells activated endothelial angiogenesis in indirect co-culture with endothelial cells (ECs), which was reversed by treatment with respective inhibitors of PAI-1 or IL-8 receptors CXCR1/2. Indirect co-culture was performed by culture of HBMECs in a conditioned medium (CM) containing the media derived from evGBMs or oxGBMs and ECGM in a ratio of 1:1. PAI-1 inhibitor tiplaxtinin (Tip, 30 µM) and CXCR1/2 inhibitor reparixin (Rep, 1 µM) or vehicle DMSO (0.1%) was added to CM, followed by EC behavior study. All data were reproduced in three independent experiments. (A) Proliferation assay in HBMEC and HUVEC. Indirect co-culture of HBMECs and HUVECs with CM derived from oxU373 and oxLN229 stimulated EC proliferation, which was completely reversed by the treatment of tiplaxtinin, not by reparixin. (B) Scratch assay in HBMEC. Left panel: images were acquired 24 h after scratching. Scale bar: 200 µm. Right panel: quantitative analysis. Culture of HBMECs with CM derived from oxU373 or oxLN229 (oxCM) significantly promoted HBMEC migration, which was reversed by the treatment of tiplaxtinin and reparixin, respectively. (C) Transwell invasion assay in HBMEC. Left panel: Representative images of invaded cells were acquired after 24 h of incubation. Scale bar: 100 µm. Right panel: quantitative analysis. Culture of HBMECs with oxCM accelerated HBMEC invasion. This effect was significantly inhibited by the treatment of reparixin but not by tiplaxtinin. (D) Tube formation assay in HBMEC. Left panel: representative images of tube formation. Scale bar: 200 µm. Right panel: quantitative analysis of branching points per field. Tube formation in HBMECs was stimulated by incubation with oxCM, which was completely diminished by both inhibitors. (E) Sprouting assay in HBMEC. Left panel: representative images of sprouting in HBMECs after 24 h of co-culture. Scale bar: 100 µm. A pronounced increase in sprouting was observed in HBMECs cultured in oxCM. Tiplaxtinin and reparixin suppressed the sprouting effect resulting from oxCM. *, p < 0.05; **, p < 0.01 and ***, p < 0.001, compared with evCM. #, p < 0.05; ##, p < 0.01 and ###, p < 0.001, compared with oxCM.

Article Snippet: To evaluate the potential angiogenic factors released from oxGBMs, an array was carried out using a human angiogenesis array kit (cat# ARY007; R&D Systems, Wiesbaden, Germany) according to the manufacturer’s protocol.

Techniques: Over Expression, Co-Culture Assay, Derivative Assay, Proliferation Assay, Wound Healing Assay, Migration, Transwell Invasion Assay, Incubation, Tube Formation Assay, Cell Culture

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 4. oxGBM-derived culture media stimulated angiogenesis on CAM, which was fully rescued by the treatment of tiplaxtinin and reparixin. CAM was incubated with the culture media derived from evU373 or oxU373 cells with or without tiplaxtinin (Tip, 30 µM) or reparixin (Rep, 1 µM) or DMSO (as a vehicle control, 0.1%) for 72 h. (A) Microscopy view of the vasculature structure on CAM. More enriched microvessel network was clearly visible in the ox group, which was significantly reduced in tiplaxtinin- and reparixin-treated CAMs. The images were acquired using a stereo microscope on ED13 (scale bar: 1 mm). Stem vessel (arrow); branched microvessel network (arrow heads). (B) Histological features of CAM after hematoxylin-eosin (H&E) staining. The CAM consists of the chorionic epithelium layer (ChE), allantoic epithelium (AE) layer, and the mesenchymal (MES) layer (arrows). Microvessel (arrowheads) density was much higher in the MES layer of ox section compared to ev section, which was clearly reduced in tiplaxtinin- and reparixin-treated sections. Scale bar: 50 µm. (C) Quantitative analysis of branching point of vessels based on microscopy images. The number of branching points and microvessels was counted by the ImageJ software (v1.1.53t) in 3 fields/CAM (n = 10 CAM/group). (D) Quantitative analysis of microvessel numbers based on H&E-stained CAM sections. Microvessel number was counted manually on H&E-stained CAMs. 10 fields/section (n = 6 sections/group). ***, p < 0.001, compared with ev. ###, p < 0.001, compared with ox.

Article Snippet: To evaluate the potential angiogenic factors released from oxGBMs, an array was carried out using a human angiogenesis array kit (cat# ARY007; R&D Systems, Wiesbaden, Germany) according to the manufacturer’s protocol.

Techniques: Derivative Assay, Incubation, Control, Microscopy, Staining, Software