vegf165  (R&D Systems)

Journal: Nature Communications

Article Title: Breast cancer remodels lymphatics in sentinel lymph nodes

doi: 10.1038/s41467-025-64981-z

Figure Lengend Snippet: a Expression of MGP in LECs after direct exposure to recombinant VEGF165 (5, 25, or 100 ng/mL), VEGF-C (5, 50, or 100 ng/mL), TGF-β (5, 25, or 100 ng/mL), or EGF (5, 25, or 100 ng/mL) are shown ( n = 4 HLECs). Data were depicted as Tukey box plots and analyzed using one-way ANOVA linear mixed models (Sidak correction) fitted separately for each parameter with group (recombinant vs control), dose and their interaction as fixed effects. b Gene expression changes in modified CM-exposed LECs are shown as determined by qPCR. CM was generated in the presence of antibodies against VEGFR3 (1 or 10 μg/mL, n = 4 HLECs), TGF-β (2 or 20 μg/mL, n = 8–10 HLECs), and EGF (1 or 10 μg/mL, n = 3–5 HLECs), compared to isotype control exposed samples and data were depicted as Tukey box plots showing relative gene changes and analyzed using a one-way ANOVA linear mixed models (Sidak) fitted separately for each parameter with group (antibody vs control) and dose and their interaction as fixed effects. c MGP expression determined by qPCR. CM generated with culture media devoid of VEGF supplement was used. Data were shown as Tukey box plots ( n = 9 HLECs). Data were analyzed using a one-way ANOVA linear mixed model (Sidak). The center line of the box plots represents the median, the box the 25th to 75th percentiles and the whiskers the inner fences. Statistics of group and dose effects are presented within the boxes; significant differences in comparison to the controls (defined as 1) are indicated by p values. Source data, non-significant p values and detailed experiment and n -numbers (biologically independent samples of cultured cells) are provided in the Source Data file.

Article Snippet: AACOCF3 (ab120350) was obtained from Abcam, fludarabine (S1491) from Seleckchem, antibodies targeting adrenomedullin (AF6108), EGF (AB-236NA), goat IgG control (AB-108-C), PDGF (AB-20-NA), rabbit IgG control (AB-105-C), and VEGF165 (AB-293-NA) from R&D Systems.

Techniques: Expressing, Recombinant, Control, Gene Expression, Modification, Generated, Comparison, Cell Culture

Journal: Molecular Cancer Research

Article Title: Attenuating Adaptive VEGF-A and IL8 Signaling Restores Durable Tumor Control in AR Antagonist–Treated Prostate Cancers

doi: 10.1158/1541-7786.MCR-21-0780

Figure Lengend Snippet: Enzalutamide treatment directly modulates tumor vasculature. LNCaP in vivo tumors ( N = 4/group) treated with enzalutamide (Enz) in the presence or absence of anti-IL8 nAb (50 μg/mL) and/or anti-VEGF nAb (100 μg/mL) for 28 days and measured time-dependent changes in ( A ) intratumoral oxygenation concentration (mmHg) and ( B ) tumor vessel density. Values shown are mean±SD. Treatment schematic is illustrated above the graph. Stereological methods were used to analyze the change in vessel density over time in all treatment groups and values were used to calculate percentage area covered by vessels. C, qRT-PCR data demonstrating detectable basal AR expression in LNCaP and HUVEC cells. Data shown are mean±SEM; N = 4 experiments. D, Representative images of prostate tumor stained for (i, ii) AR (20X and 40X), (iii, iv) CD31 (20X and 40X), and (v) hematoxylin and eosin (20X). Endothelial cells (EC) and vessels are marked by black arrows in the prostate tumor. E, Effect of 10 μmol/L Enz on in vitro tubule formation over 10 days. The number of junctions was measured using AngioSys 2.0 software. Data presented are mean±SEM of N = 8 fields of view; N = 4 experiments ( F ) Effect of 10 μmol/L Enz on viability of HUVEC cells following either 72-hour normoxic or hypoxic conditions. Data shown are mean±SEM; N = 3 experiments. G, Effect of 10 μmol/L Enz on apoptosis in HUVEC cells following either 72-hour normoxic or hypoxic conditions. Data shown are mean±SEM; N = 3 experiments. H, Effect of 10 μmol/L Enz on the colonization of PC3 cells over 5 days in the in ovo assay. Data shown are mean ± SEM of N = 17 embryos for DMSO and N = 23 embryos for the Enz. For all data, control cells were treated with equivalent volume of DMSO and statistically significant differences were determined using a Student two-tailed t test or Mann–Whitney U test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Human IL8 monoclonal antibody (RRID:AB_2249110; #MAB208), human VEGF mAb (RRID:AB_354341; #AB293NA) and isotype-matched IgG (RRID:AB_357344; #MAB002) were obtained from R&D Systems.

Techniques: In Vivo, Concentration Assay, Quantitative RT-PCR, Expressing, Staining, In Vitro, Software, In Ovo, Control, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Cancer Research

Article Title: Attenuating Adaptive VEGF-A and IL8 Signaling Restores Durable Tumor Control in AR Antagonist–Treated Prostate Cancers

doi: 10.1158/1541-7786.MCR-21-0780

Figure Lengend Snippet: Treatment-mediated hypoxia-promoted VEGF and IL8 signaling influences AR expression and activity, angiogenesis and enzalutamide response. A and B, Effect of 10 μmol/L enzalutamide (Enz) on viability of LNCaP ( A ) or C4-2B ( B ) cells under normoxia and hypoxia for 72 hours. Control cells are presented as 100% for either untreated normoxic cells (left y-axis) or cells treated with hypoxia alone (right y-axis). As a control, were treated with an equivalent volume of DMSO. Data shown are mean ± SEM of N = 3 experiments. C, Cell-cycle analysis of 10 μmol/L Enz treated LNCaP and C4–2B cells following 72 hours. Control cells were treated with an equivalent volume of DMSO. Data are mean±SEM of N = 4 experiments. D, Effect of hypoxia on expression of the AR in LNCaP cells ( top ), and AR FL and AR-V7 expression in 22Rv1 ( middle ), and CWR-R1 ( bottom ) cells. Blots shown are representative of N = 3 experiments. Equal loading was assessed using β-Actin. Relative expression was determined by densitometry using Image J software. E, Luciferase reporter assay demonstrating the effect of 2 to 24-hour hypoxia on AR transcriptional activity in LNCaP and 22Rv1 cells. Data are mean±SEM of N = 3 experiments. F, Luciferase reporter assay demonstrating the effect of hypoxia (6 hours) on AR transcriptional activity in LNCaP and 22Rv1 cells. Data are mean±SEM of N = 3 experiments. G, Immunofluorescent analysis of AR distribution in LNCaP cells cultured under normoxia or hypoxia (6 hours). Images present a merged image, DAPI staining (Blue), and AR-related fluorescence (Red). H, qRT-PCR data demonstrating detectable KLK3 (PSA) expression in LNCaP, 22Rv1, and CWRR1 cells. Data shown are mean±SEM of N = 3 experiments. For all experiments, statistical analysis was carried out using a Student two-tailed t test, Mann–Whitney U test, or 2-way ANOVA with Bonferroni post-tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Human IL8 monoclonal antibody (RRID:AB_2249110; #MAB208), human VEGF mAb (RRID:AB_354341; #AB293NA) and isotype-matched IgG (RRID:AB_357344; #MAB002) were obtained from R&D Systems.

Techniques: Expressing, Activity Assay, Control, Cell Cycle Assay, Software, Luciferase, Reporter Assay, Cell Culture, Staining, Fluorescence, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Cancer Research

Article Title: Attenuating Adaptive VEGF-A and IL8 Signaling Restores Durable Tumor Control in AR Antagonist–Treated Prostate Cancers

doi: 10.1158/1541-7786.MCR-21-0780

Figure Lengend Snippet: IL8 and VEGF signaling sustain AR pathway activation and modulate response to enzalutamide. Cells were treated with anti-IL8 nAb (5 μg/mL), anti-VEGF nAb (10 μg/mL) or the highest concentration of isotype-matched human IgG antibody. A, Effect of anti-IL8 nAb/anti-VEGF nAb on the response of hypoxic or normoxic LNCaP cells to 10 μmol/L Enazlutamide (Enz) over 72 hours. Data shown are mean ± SEM of N = 3 experiments. B, Effect of anti-IL8 nAb and/or anti-VEGF nAb on hypoxia (6 hours)-induced AR and AR-V7 expression in LNCaP and CWRR1 cells. Blots are representative of N = 3 experiments. Equal loading was assessed using GAPDH. Relative expression was determined by densitometry using Image J software. C, Effect of 10 μmol/L E (Enz) with anti-IL8 nAb (5 μg/mL), anti-VEGF nAb (10 μg/mL) or the highest concentration of isotype-matched human IgG antibody on viability of LNCaP cells under normoxia and hypoxia for 72 hours. D, Effect of VEGF (2 ng/mL) or rhIL8 (3 nmol/L) on tubule formation over 10 days. Suramin (20 μmol/L) was included as a negative control. E, Effect of CM harvested from LNCaP cells cultured in hypoxia for 24 hours, in the presence or absence of anti-IL8 nAb and/or anti-VEGF nAb, on tubule formation over 10 days. For both experiments ( D and E ), number of junctions was measured using AngioSys 2.0 software. Data are mean±SEM of N = 8 fields of view. F, Tumor growth data ( N = 5/group), obtained by measuring tumor volume every 2 days for 28 days. Treatment groups were: vehicle-only (VC); Enz (4 mg/kg); Enz (4 mg/kg) + IgG (150 μg/mL); Enz (4 mg/kg) + anti-VEGF nAb (100 μg/mL); and Enz (4 mg/mL) + anti-VEGF (100 μg/mL) and anti-IL8 (50 μg/mL) nAbs. Treatment schematic is shown above graph. Data points represent mean ± SEM. G, Average tumor weights at study completion. Values are mean ± SEM ( N = 5/group). For all experiments statistical analysis was carried out using Student two-tailed t test or Mann–Whitney U test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Human IL8 monoclonal antibody (RRID:AB_2249110; #MAB208), human VEGF mAb (RRID:AB_354341; #AB293NA) and isotype-matched IgG (RRID:AB_357344; #MAB002) were obtained from R&D Systems.

Techniques: Activation Assay, Concentration Assay, Expressing, Software, Negative Control, Cell Culture, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Cancer Research

Article Title: Attenuating Adaptive VEGF-A and IL8 Signaling Restores Durable Tumor Control in AR Antagonist–Treated Prostate Cancers

doi: 10.1158/1541-7786.MCR-21-0780

Figure Lengend Snippet: VEGF and IL8 expression is altered and plays a role in resistance in enzalutamide-resistant prostate cancer cell lines. Cells were treated with anti-IL8 nAb (5 μg/mL), anti-VEGF nAb (10 μg/mL), or the highest concentration of isotype-matched human IgG antibody. A, qRT-PCR data comparing basal expression of VEGF A and CXCL8 (IL8) expression in LNCaP-Parental (PAR), LNCaP-EnzR, CWR-R1-Par, and CWR-R1-EnzR cell lines. B, ELISA data comparing basal secretion of VEGF and IL8 in LNCaP-Par, LNCaP-EnzR, CWR-R1-Par, and CWR-R1-EnzR cells. Data shown are the mean±SEM of N = 4 experiments. C–F, Bar graphs demonstrating the effect of combined treatment with anti-IL8 nAb and anti-VEGF nAb on the response of LNCaP-Par, LNCaP-EnzR, CWR-R1-Par, and CWR-R1-EnzR cells to 10 μmol/L Enz over 72 hours in ( C and D ) normoxia and ( E and F ) hypoxia. All experiments data represented as the mean ± SEM of N = 3 experiments, unless otherwise stated and statistical analysis was carried out using a Mann-Whitney U test: *, P < 0.05; **, P < 0.01.

Article Snippet: Human IL8 monoclonal antibody (RRID:AB_2249110; #MAB208), human VEGF mAb (RRID:AB_354341; #AB293NA) and isotype-matched IgG (RRID:AB_357344; #MAB002) were obtained from R&D Systems.

Techniques: Expressing, Concentration Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Molecular Cancer Research

Article Title: Attenuating Adaptive VEGF-A and IL8 Signaling Restores Durable Tumor Control in AR Antagonist–Treated Prostate Cancers

doi: 10.1158/1541-7786.MCR-21-0780

Figure Lengend Snippet: Inhibition of VEGF and IL8 enhances tumor sensitivity to enzalutamide in vivo. For all experiments, male Balb /c SCID mice bearing tumors of 100 to 150 mm 3 were assigned to the following treatment groups for 28 days: Vehicle; enzalutamide (Enz; 4 mg/kg) + IgG control (150 μg/mL); Vehicle + anti-VEGF nAb (100 μg/mL) and anti-IL8 (50 μg/mL) nAbs; and Enz (4 mg/mL) + anti-VEGF (100 μg/mL), and anti-IL8 (50 μg/mL) nAbs. A and B, Tumor growth data, obtained by measuring ( A ) LNCaP-PAR and ( B ) LNCaP-EnzR tumor volume every 2 days. The treatment schematic is shown above the graph. The data points represent the mean ± SD ( N = 8/group). C and D, Normalized bodyweight (at end of treatment) of mice with ( C ) LNCaP-PAR and ( D ) LNCaP-EnzR tumors treated with Enz alone or in combination with anti-VEGF nAb and anti-IL8 nAb. Values shown are mean ± SD ( N = 8/group). E and F, Intratumoral oxygenation concentration (mmHg) in ( E ) LNCaP-Par and (F ) LNCaP-EnzR in vivo tumors ( N = 4/group) treated with Enz alone or in combination with anti-VEGF nAb and anti-IL8 nAb for 29 days and measured time-dependent changes. For all experiments, statistical analysis was carried out using a 2-way ANOVA with Tukey post-hoc test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Human IL8 monoclonal antibody (RRID:AB_2249110; #MAB208), human VEGF mAb (RRID:AB_354341; #AB293NA) and isotype-matched IgG (RRID:AB_357344; #MAB002) were obtained from R&D Systems.

Techniques: Inhibition, In Vivo, Control, Concentration Assay

Journal: Scientific Reports

Article Title: Preferential stimulation of melanocytes by M2 macrophages to produce melanin through vascular endothelial growth factor

doi: 10.1038/s41598-022-08163-7

Figure Lengend Snippet: Neutralizing of hVEGF in hiMels cocultured with M2 macrophages. ( a ) Schematic diagram showing 5-day coculture of hiMels and M2 macrophages with or without 120 ng/mL human VEGF165 Antibody. ( b ) Melanin content of hiMels measured on day 5 coculture. ( c ) Pigmentation of cocultured hiMels with or without VEGF antibody by L-DOPA staining. Scale bar indicates 100 µm. ( d ) Quantitative measurements of L-DOPA staining area in cocultured hiMels by ImageJ software. ( e ) Quantification of tyrosinase activity in cocultured hiMels. ( f ) Quantitation of expression of pigmentation marker (MITF, TYRP1, TYR, DCT, and MLANA) in hiMels after 5 days of coculture. Relative gene expression values were calculated using the delta cycle-threshold (ΔCt) method. ( g ) Protein expression level of MITF, TYR, and TRP1 in hiMels. GAPDH was used as a loading control. ( h ) Quantification of western blotting using ImageJ software. * p < 0.05, ** p < 0.01, *** p < 0.001 ( t -test), # p < 0.05, ## p < 0.01, ### p < 0.001 vs. M2 (one-way analysis of variance) indicate statistical significance. MΦ macrophage, hiMels human induced pluripotent stem cell (hiPSCs)-derived melanocytes, VEGF anti human VEGF165 antibody, L-DOPA 3,4-dihydroxyphenylalanine, M2 anti cocultured with M2 and VEGF antibody.

Article Snippet: The cells were incubated in a combination of media comprised of 70% full Mel medium and 30% RPMI medium supplemented with CHIR99021 (3 mM), BMP4 (25 ng/mL), and EDN3 (100 nM), with or without human VEGF165 antibody (120 ng/mL; AF-293-SP, R&D Systems) at 37 °C and 5% CO 2 .

Techniques: Staining, Software, Activity Assay, Quantitation Assay, Expressing, Marker, Gene Expression, Control, Western Blot, Derivative Assay