Journal: Cell reports

Article Title: Activation of Wnt/β-catenin signaling by Zeb1 in endothelial progenitors induces vascular quiescence entry.

doi: 10.1016/j.celrep.2022.111694

Figure Lengend Snippet: Figure 3. Deletion of Zeb1 results in vessel overgrowth and severe deformity (A) Hematoxylin and eosin (H&E) stain shows sites of vessel enlargement and hemorrhaging in Cdh5-Cre;Zeb1fl/flretina. Scale bar, 50 mm. (B) Images of P16 retinal cup indicate retinal vessel leakage in Zeb1ECKO. Scale bar, 5 mm. (C) 3D construction of tissue-cleared whole retina from day 20 control (left) and Zeb1ECKO (right) mice. Alexa Fluor 647-conjugated isolectin (IB4) was injected intravenously before sacrifice to label functional circulatory vasculature. Mutant retina displayed loss of hierarchical patterning, vessel dilation, and areas of obstructed flow (yellow arrow). Scale bar, 2 mm. (D and E) Representative image of P16 retina vasculature from control (D) and Zeb1ECKO (E) showing grossly enlarged vessels with accumulated ECs (indicated as ERG+) as well as sites of arteriovenous malformation (AVM; indicated by yellow arrows). Scale bar, 100 mm. (F and G) Representative image of P16 retina indicates the formation of numerous aneurysms in Zeb1ECKO mutant. Scale bar, 100 mm. (H and I) Cross section of P16 eyeball shows disarrayed vascular patterning and hyperplasia in Zeb1ECKO retina at both superficial and deep vascular layers. Scale bar, 75 mm. (J–M) Whole-mount retina staining for FOXO1 (white), VE-cadherin (Cdh5, magenta), and IB4 (green) in P6.5 (J and K) and P10 (L and M) retina. Zoomed-in view depicts Cdh5 and FOXO1 signal in yellow dash-lined band. Scale bar, 200 mm (J and K); 500 mm (L and M).

Article Snippet: The primary antibodies used in immunofluorescence were rat anti-mouse Cell Reports 41, 111694, November 22, 2022 e4 CD31 (BD Bioscience, clone # MEC 13.3,1:100), rat anti-mouse CD144 (BD Biosciences, clone# MEC13.3, 1:100), rabbit anti-ERG antibody (Abcam, Cat # ab92513, 1:200), rabbit anti-FOXO1 antibody (CST, Cat # 2880S,1:200), rabbit anti-mouse Keratin5 (BioLegend, Cat # 905504, 1:500), mouse anti-E-Cadherin (BD Biosciences, Cat# 610182, 1:500), rat anti-mouse PLVAP antibody(BD PharmingenTM, Cat# 553849, 1:100), Isolectin IB4-647 (Thermo Fisher, Cat #l32450, 1:100), Isolectin IB4-488 (Thermo Fisher, Cat# l21411, 1:100), rabbit anti-Ki67 (Abcam, Cat# ab15580, 1:1000).

Techniques: Staining, Control, Injection, Functional Assay, Mutagenesis

Journal: Cell reports

Article Title: Activation of Wnt/β-catenin signaling by Zeb1 in endothelial progenitors induces vascular quiescence entry.

doi: 10.1016/j.celrep.2022.111694

Figure Lengend Snippet: Figure 6. Administration of Wnt agonist NGS ameliorates Zeb1ECKO vascular defects (A) Timeline depicting experimental procedures and tissue collection. (B and C) Activation of Wnt/b-catenin signaling by NGS was validated both by qPCR on Wnt target Axin2 (B) and through western blotting detecting for Lrp6 phosphorylation (C). (D and E) Zeb1ECKO retina whole-mount staining shows vasculature after treatment with saline (D) or NGS (E). Scale bar, 1 mm. (F–H) FOXO1 activation is absent in saline-injected Zeb1ECKO retina vasculature (F), while NGS injection restores FOXO1 nuclear activation (G) and resumed the process of vessel remodeling with regression sites indicated by yellow arrows (H). Scale bar, 50 mm. (I and J) Whole-mount image of corneolimbal region injected with either saline (I) or NGS (J). NGS treatment ameliorates vascular hyperplasia in Zeb1ECKO

Article Snippet: The primary antibodies used in immunofluorescence were rat anti-mouse Cell Reports 41, 111694, November 22, 2022 e4 CD31 (BD Bioscience, clone # MEC 13.3,1:100), rat anti-mouse CD144 (BD Biosciences, clone# MEC13.3, 1:100), rabbit anti-ERG antibody (Abcam, Cat # ab92513, 1:200), rabbit anti-FOXO1 antibody (CST, Cat # 2880S,1:200), rabbit anti-mouse Keratin5 (BioLegend, Cat # 905504, 1:500), mouse anti-E-Cadherin (BD Biosciences, Cat# 610182, 1:500), rat anti-mouse PLVAP antibody(BD PharmingenTM, Cat# 553849, 1:100), Isolectin IB4-647 (Thermo Fisher, Cat #l32450, 1:100), Isolectin IB4-488 (Thermo Fisher, Cat# l21411, 1:100), rabbit anti-Ki67 (Abcam, Cat# ab15580, 1:1000).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Staining, Saline, Injection

Journal: Frontiers in Pharmacology

Article Title: A Novel Small Molecule GDNF Receptor RET Agonist, BT13, Promotes Neurite Growth from Sensory Neurons in Vitro and Attenuates Experimental Neuropathy in the Rat

doi: 10.3389/fphar.2017.00365

Figure Lengend Snippet: BT13 reverses spinal nerve ligation-induced changes in expression of nociception-related neuronal markers. Samples were collected on 12th day post-surgery after behavioral analysis. Immunohistochemical stainings of DRG paraffin sections with pErk, IB4, CGRP, NpY, pS6 (A) . Quantification of IHC data (B) . Quantifications of marker-specific immunopositive cells was made for two sections per ganglia, normalized to the total number of PGP9.5-positive cells, averaged, and converted to percentage from contralateral side. Arrows indicate neurons that were counted as marker-specific. The data for 3–6 animals per group were subjected to statistical analysis. Results are presented as mean ± SEM . Scale bar—100 μm. * P < 0.05, ** P < 0.01, t -test.

Article Snippet: Sections were deparaffinized, subjected to citrate or basic antigen-retrieval procedure and probed with IB4-Alexa 488 [1:200; IB4-Alexa488, Cat# IB4 (isolectin Griffonia simplicifolia IB4) RRID:AB_2314664 ] and antibodies against calcitonin gene-related peptide (CGRP; 1:10,000, Peninsula Laboratory, Cat# T-4032 RRID:AB_2313775 ), NPY (1:10,000, Peninsula Laboratory, Cat# T-4070.0050 RRID:AB_518504 ), pErk1/2 (1:300, Cell Signaling, Cat# 4370L RRID:AB_2297462 ), pS6 (1:300, Cell Signaling, Cat# 4858L RRID:AB_1031194 ) or RET [1:100, kind gift of Dr. E. Kramer, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany (Meka et al., )] and corresponding secondary antibodies conjugated with fluorophores (Donkey Anti-Rabbit Alexa Fluor 647 conjugate, Cat# A31573 RRID:AB_162544 ; Donkey anti-Mouse Alexa Fluor 647 conjugate, Cat# A-31571 RRID:AB_2536181 ; Goat anti-Guinea Pig, Alexa Fluor 568 conjugate, Cat# A-11075 RRID:AB_2534119 , Thermo Fisher Scientific, USA) or horse radish peroxidase (VECTASTAIN Elite ABC-Peroxidase Kit antibody, Vector Laboratories, CA, USA, Cat# PK-6101 RRID:AB_2336820 ).

Techniques: Ligation, Expressing, Immunohistochemical staining, Marker