recombinant zebrafish vegf165  (R&D Systems)

Journal: Developmental cell

Article Title: Macrophages trigger cardiomyocyte proliferation by increasing epicardial vegfaa expression during larval zebrafish heart regeneration.

doi: 10.1016/j.devcel.2022.05.014

Figure Lengend Snippet: Figure 7. Vegfaa drives cardiomyocyte proliferation by endocardial notch signaling (A) LSFM images of Tg(myl7:h2b-GFP) larvae at 24 hpi treated with PBS 0.1% BSA or zfVegfaa 0.1% BSA injection. (B) Ventricular cardiomyocyte number inTg(myl7:h2b-GFP) larvae at 24and 48hpi treated with PBS 0.1% BSA or zfVegfaa 0.1% BSA injection, n = 20. Unpaired t test. (C) Images of injured ventricles from Tg(myl7:h2b-GFP) larvae, EdU stained and bathed in vehicle or AV951, imaged at 48 hpi. Non-myocardial EdU signal is excluded post-acquisitionally. (D) Percentage of EdU+ cardiomyocyte nuclei from uninjured and injured ventricles from Tg(myl7:h2b-GFP) larvae, EdU stained and bathed in vehicle or AV951, n = 13–36. Unpaired t test. (E) Images of injured Tg(myl7:nlsDsRed) larvae treated with vehicle or DAPT, acquired at 48 hpi by LSFM. (F) Ventricular cardiomyocyte number in uninjured and injured Tg(myl7:h2b-GFP) larvae at 48 hpi treated with vehicle or DAPT, n = 24–40. Unpaired t test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Phenylthiourea Thermo Fisher Scientific Cat #L06690.09 Tricaine methanesulfonate Sigma Aldrich Cat #E10521 Metronidazole Thermo Fisher Scientific Cat #210340050 DMSO Sigma Aldrich Cat #20-139 Propidium iodide Thermo Fisher Scientific Cat #P1304MP zfIFN-g-rel (IFN-1.1) Kingfisher Biotech Cat #RP1058Z-025 zfVegfaa Kingfisher Biotech Cat # RP1040Z-025 Pacific blue 500kDa dextran Fina Biosolutions https://www.finabio.net/ (custom made upon request) AV-951 Stratech Scientific Cat #A2251-APE DAPT Cambridge Bioscience Cat #CAY13197-10 AG-1478 Cambridge Bioscience Cat #CAY10010244-10 Critical commercial assays RedTaq ReadyMix Sigma Aldrich Cat #R2523 ApopTag Red In situ kit MilliporeSigma Cat #S7165 EdU Imaging Kit with Alexa Fluor 594 Invitrogen Cat #C10339 RNeasy Plus Micro Kit Qiagen Cat #74034 Deposited data RNA-seq data This paper (Supplementary file 1) Raw data are publicly available at Array express: E-MTAB-10860.

Techniques: Injection, Staining

Journal: Developmental Cell

Article Title: Macrophages trigger cardiomyocyte proliferation by increasing epicardial vegfaa expression during larval zebrafish heart regeneration

doi: 10.1016/j.devcel.2022.05.014

Figure Lengend Snippet:

Article Snippet: zfVegfaa , Kingfisher Biotech , Cat # RP1040Z-025.

Techniques: Recombinant, In Situ, Imaging, Software

Journal: Developmental Cell

Article Title: Macrophages trigger cardiomyocyte proliferation by increasing epicardial vegfaa expression during larval zebrafish heart regeneration

doi: 10.1016/j.devcel.2022.05.014

Figure Lengend Snippet: Vegfaa drives cardiomyocyte proliferation by endocardial notch signaling (A) LSFM images of Tg(myl7:h2b-GFP) larvae at 24 hpi treated with PBS 0.1% BSA or zfVegfaa 0.1% BSA injection. (B) Ventricular cardiomyocyte number in Tg(myl7:h2b-GFP) larvae at 24 and 48 hpi treated with PBS 0.1% BSA or zfVegfaa 0.1% BSA injection, n = 20. Unpaired t test. (C) Images of injured ventricles from Tg(myl7:h2b-GFP) larvae, EdU stained and bathed in vehicle or AV951, imaged at 48 hpi. Non-myocardial EdU signal is excluded post-acquisitionally. (D) Percentage of EdU+ cardiomyocyte nuclei from uninjured and injured ventricles from Tg(myl7:h2b-GFP) larvae, EdU stained and bathed in vehicle or AV951, n = 13–36. Unpaired t test. (E) Images of injured Tg(myl7:nlsDsRed) larvae treated with vehicle or DAPT, acquired at 48 hpi by LSFM. (F) Ventricular cardiomyocyte number in uninjured and injured Tg(myl7:h2b-GFP) larvae at 48 hpi treated with vehicle or DAPT, n = 24–40. Unpaired t test. (G) Images of injured Tg(myl7:nlsDsRed) larvae treated with vehicle or AG1478, acquired at 48 hpi by LSFM. (H) Ventricular cardiomyocyte number in uninjured and injured Tg(myl7:h2b-GFP) larvae at 48 hpi treated with vehicle or AG1478, n = 24. Unpaired t test. (I) Treatment strategy for the injection of uninjured larvae with zfVegfaa and continuous bathing in AG1478 solution. (J) Hypothesized signaling pathway active in uninjured and injured larval hearts driving cardiomyocyte proliferation. (K) LSFM-acquired z plane showing notch expression colocalizing with endocardium in Tg(Tp1:venus-PEST;kdrl:hsa.HRAS-mCherry) , abbreviated in the figure to Tg(Tp1:venus-PEST;kdrl:mCherry) . AG1478 abbreviated to AG; white box, zoom panel. (L) Proportion of larvae with notch+ endocardium at 6, 24, and 48 hpt following zfVegfaa injection and bathing in AG1478, n = 28. Fisher’s exact test. (M) Treatment strategy for the lasering of larvae and continuous bathing in AG1478 solution. (N) Representative z plane images of uninjured, injured, and injured AG-treated ventricles from Tg(tp1:venus-PEST) larvae at 48 hpi. BA, bulbous arteriosus; AVV, atrioventricular valve; white arrowheads, laterally inhibited cardiomyocytes; cyan arrowheads, notch+ endocardium; cyan box, zoom panel. Fisher’s exact test. (O) Proportion of larvae with notch+ endocardium at 6, 24, and 48 hpt following laser injury and bathing in AG1478, n = 18. (P) Cardiomyocyte number at 48 hpi following injection with recombinant Vegfaa and continuous bathing in DAPT or AG1478, n = 22–25. One-way ANOVA followed by Holms-Sidak’s multiple comparison post-hoc tests. All images are maximum intensity projections of 3D LSFM stacks unless otherwise stated. Scale bars, 50 μm. Data are represented as mean ± SEM, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001.

Article Snippet: zfVegfaa , Kingfisher Biotech , Cat # RP1040Z-025.

Techniques: Injection, Staining, Expressing, Recombinant, Comparison

Journal: Developmental Cell

Article Title: Macrophages trigger cardiomyocyte proliferation by increasing epicardial vegfaa expression during larval zebrafish heart regeneration

doi: 10.1016/j.devcel.2022.05.014

Figure Lengend Snippet:

Article Snippet: zfVegfaa , Kingfisher Biotech , Cat # RP1040Z-025.

Techniques: Recombinant, In Situ, Imaging, Software

Journal: mSphere

Article Title: The Passenger Domain of Bartonella bacilliformis BafA Promotes Endothelial Cell Angiogenesis via the VEGF Receptor Signaling Pathway

doi: 10.1128/msphere.00081-22

Figure Lengend Snippet: BafA Bba upregulates the VEGFR2-ERK signaling pathway. (A) Phosphorylation of VEGFR2 and ERK1/2 in HUVECs stimulated with vehicle control (HBSS), VEGF-A 165 (20 ng/mL), BafA Bhe (200 ng/mL), or BafA Bba (200 ng/mL) for the indicated times. Western blot analysis was performed using antibody against phosphorylated (p-) and total VEGFR2 or ERK1/2. (B) Quantification of BafA Bba -induced phosphorylation of ERK1/2. The phosphorylation ratio was normalized to total ERK1/2, with the value for untreated cells (time zero) set as 1. Bars show means and SD ( n = 3 biological replicates; circles). Statistical significance was determined using one-way ANOVA with Dunnett’s multiple-comparison test (*, P < 0.05).

Article Snippet: The interaction of recombinant Fc-tagged VEGFR2-ECD (Sino Biological, Beijing, China) with VEGF-A 165 , BafA Bhe , or BafA Bba was monitored by SPR using a Biacore 8K (Cytiva) performed at 25°C in single-cycle mode.

Techniques: Control, Western Blot, Comparison

Journal: mSphere

Article Title: The Passenger Domain of Bartonella bacilliformis BafA Promotes Endothelial Cell Angiogenesis via the VEGF Receptor Signaling Pathway

doi: 10.1128/msphere.00081-22

Figure Lengend Snippet: Effect of VEGFR2 inhibition on BafA Bba activity and binding profiles of VEGFR2 with BafAs. (A) Inhibitory effect of anti-VEGFR2 antibody on the mitogenic activity of BafA Bba . HUVECs were pretreated with PBS, human normal IgG (3 μg/mL), anti-VEGF antibody (bevacizumab, 3 μg/mL), or anti-VEGFR2 antibody (ramucirumab, 3 μg/mL), before stimulation with vehicle control (HBSS), VEGF-A 165 (20 ng/mL), or BafA Bba (50 ng/mL). Bars show means and SD ( n = 8 biological replicates; circles). (B) Expression of VEGFR2 in HUVECs transfected with noncoding small interfering RNA (si-NC) or siRNAs coding for VEGFR2 (si-VEGFR2). VEGFR2 mRNA relative expression was quantified by qRT-PCR. Bars show means and SD ( n = 3 biological replicates). (C) Cell proliferation of siRNA-transfected HUVECs stimulated with control (HBSS), VEGF-A 165 (20 ng/mL), or BafA Bba (100 ng/mL). Bars show means and SD ( n = 8 biological replicates; circles). (D) Binding profiles of VEGF-A 165 , BafA Bhe , and BafA Bba with VEGFR2, characterized by surface plasmon resonance analysis. The Fc-tagged VEGFR2 was captured on protein A sensor chips and subsequently tested for binding with gradient concentrations (1.25, 2.5, 5, 10, and 20 nM) of VEGF-A 165 , BafA Bhe , or BafA Bba with the binding profiles shown. The K D values shown are means and SD from three independent experiments. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple-comparison test (A and C) or a two-tailed unpaired Student's t test (B). ns, not significant; ***, P < 0.001.

Article Snippet: The interaction of recombinant Fc-tagged VEGFR2-ECD (Sino Biological, Beijing, China) with VEGF-A 165 , BafA Bhe , or BafA Bba was monitored by SPR using a Biacore 8K (Cytiva) performed at 25°C in single-cycle mode.

Techniques: Inhibition, Activity Assay, Binding Assay, Control, Expressing, Transfection, Small Interfering RNA, Quantitative RT-PCR, SPR Assay, Comparison, Two Tailed Test