Journal: Journal of Cell Science

Article Title: c-Src-induced vascular malformations require localised matrix degradation at focal adhesions

doi: 10.1242/jcs.262101

Figure Lengend Snippet: Constitutively active c-Src increased VE-cadherin phosphorylation and decreases 3D cell migration. (A) Representative images of western blots of VEGFA-induced VEGFR2 and VE-cadherin phosphorylation in confluent monolayers of HUVECs transduced with mScarlet-tagged c-Src WT, mutants (CA or DN) or control (Ctrl; empty vector mScarlet) and starved overnight in serum-reduced medium before being treated with 100 ng/ml VEGFA for 0 min, 5 min or 15 min. Activation of VEGFR2 and VE-cadherin (VEC) are shown by phosphorylation (p) on indicated tyrosine sites. GAPDH was used as loading control. Full blots with all respective loading controls can be found in Fig. S8 . (B) Quantification of VEGFR2 phosphorylation at Tyr(Y)951 confirms receptor activation with a peak at 5 min. Ratio of phosphorylated VEGFR2 (Y951) to total VEGFR2 corrected to loading control (GAPDH). (C) Quantification of VE-cadherin phosphorylation at Y658 relative to total VE-cadherin corrected for loading control (GAPDH) and normalised to 0 min. (D) Quantification of VE-cadherin phosphorylation at Y731 relative to total VE-cadherin corrected for loading control (GAPDH) and normalised to 0 min. n =3–4 independent experiments. (E) HUVECs transduced as in A were grown in a 5 mg/ml fibrin gel bead sprouting assay for 5 days before live imaging at 30 min intervals over 72 h (representative images of 0 and 72 h are shown). (F,G) Quantification of total distance from the bead within 72 h (F) and average velocity of the sprouting front (G). n =3 independent experiments. using Kruskal–Wallis test with Dunn's multiple comparisons. All data are represented as mean±s.e.m. with individual data point indicated and colours represent independent experiments. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 [two-way ANOVA with Tukey's multiple comparisons (B–D) or Kruskal–Wallis test with Dunn's multiple comparisons (F,G)]. a.u., arbitrary units.

Article Snippet: The following antibodies and dyes were used: rabbit anti-phospho-c-Src (Y416) (Invitrogen, 44660G, 1:100 for immunocytochemistry (ICC)], rabbit anti-phospho-VE-cadherin (Y658) [Invitrogen, 44-1144G, 1:1000 for western blotting (WB)], rabbit anti- phospho-VE-cadherin (Y731) (Invitrogen, 44-1145G, 1:500 for WB), rabbit anti-phospho-paxillin (Y118) (Invitrogen, 44-722G, 1:1000 for WB, 1:200 for ICC), mouse anti-c-Src GD11 (Millipore, 05-184, 1:1000 for WB, 1:200 for ICC), goat anti-VE-cadherin (R&D systems, AF938, 1:2000 for WB), goat anti-VEGFR2 (R&D systems, AF357, 1:1000 for WB), mouse anti-VE-cadherin-Alexa Fluor 647 (BD Biosciences, 561567, 1:250 for ICC), mouse anti-phospho-FAK (Y397) (BD Biosciences, 611806, 1:200 for ICC, 1:1000 for WB), mouse anti-fibronectin (BD Biosciences, 610077, 1:200 for ICC), rabbit anti-phosho-paxillin (Y118) (Abcam, AB4833, 1:100 for ICC), rabbit anti-Ki-67 (Abcam, ab15580, 1:200 for ICC), rabbit anti-paxillin (Cell Signaling, 2542, 1:1000 for WB), rabbit anti-phospho-FAK (Y576) (Cell Signaling, 3281, 1:1000 for WB), rabbit anti- phospho-VEGFR2 (Y951) (Cell Signaling, 2471, 1:1000 for WB), rabbit anti-GAPDH (Cell Signaling, 2118, 1:5000 for WB), rabbit anti-DLL4 (Cell Signaling, 2589, 1:500 for WB), mouse anti-FAK (Santa Cruz Biotechnology, sc-271126, 1:1000 for WB), phalloidin (conjugated to Alexa Fluor 488 and 670, Cytoskeleton Jomar, PHDG1-A, PHDN1-A, 1:500 for ICC), rabbit anti-cleaved caspase 3 (D175) (Cell Signaling, 1:200 for ICC) and the Click-iT EdU Cell Proliferation kit Alexa Fluor 647 dye (Invitrogen, C10340, 10 μM for ICC).

Techniques: Phospho-proteomics, Migration, Western Blot, Transduction, Control, Plasmid Preparation, Activation Assay, Imaging

Journal: The FASEB Journal

Article Title: Novel engineered, membrane‐tethered VEGF‐A variants promote formation of filopodia, proliferation, survival, and cord or tube formation by endothelial cells via persistent VEGFR2/ERK signaling and activation of CDC42/ROCK pathways

doi: 10.1096/fj.202100448rr

Figure Lengend Snippet: FIGURE 1 Transfected or transduced eVEGF-38, eVEGF-53, and VEGF189 are expressed in hRECs and activate VEGFR2 and its downstream signaling factors in a sustained manner. (A) A schematic of the engineered VEGF121 (eVEGF, top) expression construct and VEGF189 (bottom) in the AAV2 vector. For the engineered VEGF variants, a VEGF121 dimeric expression cassette was oriented head to tail. SP, signal peptide; ITR, inverted terminal repeat of the AAV vector; CMV, promoter; Myc, Myc epitope tag, GPI, signal for a glycophosphatidylinositol anchor. (B) Primary hRECs were transfected with DNA plasmids for eVEGF-38, eVEGF-53, VEGF189, or GFP control, then expression of the constructs was determined at 1–4 days by semi-quantitative real-time qPCR using primer sets specific to each construct. ***p < .001 compared to one day by unpaired two-tailed t-test, n = three independent experiments. As expected, the cells transfected with GFP did not express any of the VEGF constructs, so these data were not plotted in the graph. (C) Western blot analysis of transgene expression using anti-myc tag 1–4 days after plasmid transfection of hRECs with eVEGF-38, eVEGF-53, or VEGF189. **p < .05, ***p < .001 compared to one day for each group by unpaired two-tailed t-test, n = three independent experiments. (D) Western blot analysis of transgene expression using anti-myc tag antibody on whole cell extracts (WCE) and conditioned medium (CM) 3 days after transduction of hRECs with AAV2-eVEGF-38, AAV2-eVEGF-53, AAV2-VEGF189, or AAV2-GFP. ***p < .001 comparing levels in WCE and CM using an unpaired two-tailed t-test, n = three independent experiments. (E) Primary hRECs were transfected with DNA plasmids for eVEGF-38, eVEGF-53, VEGF189, or GFP control, then expression Dll4, and endogenous VEGF was determined at 1–4 days by semi-quantitative real-time qPCR. **p < .01 compared to the GFP control by unpaired two-tailed t-test, n = three independent experiments. (F) Western blot analysis for phosphorylated versus total levels of VEGFR2 and downstream signaling proteins at 24 and 48 h following transfection of hRECs with eVEGF-38, eVEGF-53, or VEGF189. Total cell lysates from the transfected hRECs were subjected to western blot analysis with antibodies against phospho- (p)-VEGFR2, total VEGFR2, p-AKT, total AKT, pERK1/2, total ERK1/2, p-p38, total p38, and alpha tubulin (left). The protein levels were normalized to tubulin, then the ratios of phosphorylated protein to total protein were compared (right). *p < .05, **p < .01, ***p < .001 by unpaired two-tailed t-test compared to the GFP control, n = three independent experiments. All data = mean ± SEM

Article Snippet: Antibodies against phospho- VEGFR2 (Y1175), VEGFR2, Akt, phospho- Akt (S473), p38 MAPK, phospho- p38 MAPK, p44/42 MAPK (Erk1/2), phospho- p44/42 MAPK (Erk1/2), alpha- tubulin, and myc epitope tag were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Transfection, Expressing, Construct, Plasmid Preparation, Control, Two Tailed Test, Western Blot, Transduction

Journal: The FASEB Journal

Article Title: Novel engineered, membrane‐tethered VEGF‐A variants promote formation of filopodia, proliferation, survival, and cord or tube formation by endothelial cells via persistent VEGFR2/ERK signaling and activation of CDC42/ROCK pathways

doi: 10.1096/fj.202100448rr

Figure Lengend Snippet: FIGURE 3 Expression of membrane-tethered eVEGF-38 and eVEGF-53, but not VEGF189, induces and sustains formation of elongated filopodia in hRECs. (A) Representative images of immunocytochemistry of hRECs for eVEGF-38, eVEGF-53, VEGF189 (anti-myc, green) and GFP-Ctr (auto-fluorescence, green) expression at 12 and 72 h after transient transfection. Representative elongated, filopodia-like membrane extensions are marked with white arrows). Middle row: zoomed-in boxes from the top row (12 h) showing filopodial structures at higher magnification. Scale bar = 10 µm. (B) Correlative scanning electron microscopy for eVEGF-38-transfected hRECs showing the extended filopodial structures on an eVEGF-38 expressing cell (anti-Myc, green) and short filopodia on a cell negative for eVEGF expression. The filopodia are highlighted in yellow for both cells. Scale bar = 100 µm for the middle panel, 10 µm for the right panels. (C) Representative images showing immunocytochemistry for transgenes (anti-Myc or GFP, green) and phalloidin (red) 24 h after transfection of hRECs with eVEGF-38, eVEGF-53, VEGF189 or GFP-Ctr. Elongated filopodia are indicated by the white arrows. Bottom row: zoomed-in boxes showing the filopodia at higher magnification. Scale bar = 10 µm. (D) Representative images showing immunocytochemistry for transgenes (anti-myc or GFP, green) and ezrin (red) 24 h after transfection with eVEGF-38, eVEGF-53, VEGF189 or GFP-Ctr. Elongated filopodia are indicated by the white arrows and magnified in the boxes. Scale bar = 10 µm. (E) Representative images showing immunocytochemistry for transgenes (anti-Myc or GFP, green) and VEGFR2 (red) 24 h after transfection of hRECS with eVEGF-38, eVEGF-53, VEGF189, or GFP-Ctr. Elongated filopodia are indicated by the white arrows, and co-localization at the cell surface is indicated by the arrowheads. Scale bar = 10 µm. (F) Morphometric quantification of average number of filopodia per cell and average filopodial length per cell using phalloidin- positive filopodia. Data = mean ± SEM ***p < .001 compared to the GFP control by unpaired two-tailed t-test, n = 15 cells/group from three independent experiments

Article Snippet: Antibodies against phospho- VEGFR2 (Y1175), VEGFR2, Akt, phospho- Akt (S473), p38 MAPK, phospho- p38 MAPK, p44/42 MAPK (Erk1/2), phospho- p44/42 MAPK (Erk1/2), alpha- tubulin, and myc epitope tag were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing, Membrane, Immunocytochemistry, Fluorescence, Transfection, Electron Microscopy, Control, Two Tailed Test

Journal: The FASEB Journal

Article Title: Novel engineered, membrane‐tethered VEGF‐A variants promote formation of filopodia, proliferation, survival, and cord or tube formation by endothelial cells via persistent VEGFR2/ERK signaling and activation of CDC42/ROCK pathways

doi: 10.1096/fj.202100448rr

Figure Lengend Snippet: FIGURE 4 VEGFR2 signaling via MAPK/ERK is required for eVEGF-induced formation of elongated filopodia in hRECs, and neither the VEGFR2 activation nor the formation of elongated filopodia is inhibited by aflibercept (Eylea). (A) Western blot analysis of VEGFR2 phosphorylation levels with and without sunitinib malate treatment (1 μM) in hRECs transfected with eVEGF-38 or eVEGF-53 (anti-myc tag antibody). ***p < .001 by unpaired two-tailed, n = three independent experiments. (B) Top panels, representative immunocytochemistry images of filopodia (white arrows) in hRECs expressing membrane-tethered eVEGF-38 or eVEGF-53 (anti-myc, green) with or without the VEGFR2 inhibitor sunitinib malate (1 μM), the MAPK/ERK inhibitor U0126 (10 μM), the p38 MAPK inhibitor SB203580 (10 μM) or the PI3K/AKT inhibitor LY294002 (10 μM). Scale bar = 10 µm. Bottom graphs, quantification of the average number of filopodia per cell and the average length of the three longest filopodia per cell. ***p < .001 by unpaired two-tailed t-test, N = 15 cells/group from three independent experiments. (C) Western blot analysis of phospho-VEGFR2, VEGFR2, alpha-tubulin and the eVEGFs in the presence or absence of Aflibercept (0.1 μg/ml) 24 h after transduction with eVEGF-38/AAV2, eVEGF-53/AAV2, or VEGF189/AAV. Densitometric analysis was used to provide the phospho-VEGFR2/total VEGFR2 levels normalized to the corresponding untreated control, shown as mean ± SEM. *p < .05 by two-tailed unpaired t-test, n = three independent experiments. (D) Left, immunocytochemistry showing eVEGF expression (anti-Myc tag, green) and filopodia formation 24 h after transduction with eVEGF-38/AAV or eVEGF-53/AAV, with or without Aflibercept treatment (0.1 μg/ml).20 Scale bar = 20 µm. Right, average number of filopodia per cell and average filopodial length are displayed as mean ± SEM, n = 15 cells/group from three independent experiments. NS = not significant by two-tailed unpaired t-test

Article Snippet: Antibodies against phospho- VEGFR2 (Y1175), VEGFR2, Akt, phospho- Akt (S473), p38 MAPK, phospho- p38 MAPK, p44/42 MAPK (Erk1/2), phospho- p44/42 MAPK (Erk1/2), alpha- tubulin, and myc epitope tag were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Transfection, Two Tailed Test, Immunocytochemistry, Expressing, Membrane, Transduction, Control

Journal: The FASEB Journal

Article Title: Novel engineered, membrane‐tethered VEGF‐A variants promote formation of filopodia, proliferation, survival, and cord or tube formation by endothelial cells via persistent VEGFR2/ERK signaling and activation of CDC42/ROCK pathways

doi: 10.1096/fj.202100448rr

Figure Lengend Snippet: FIGURE 6 NRP1 knockdown does not affect VEGFR2 activation nor formation of elongated filopodia in hRECS expressing eVEGFs. (A) Left panels, representative images of western blots showing VEGFR2 phosphorylation with and without NRP1 knockdown in hRECs transfected with eVEGF-38, eVEGF-53, VEGF189, or GFP control. The hRECs were transfected with siNrp1 or control siRNA (scramble) for 24 h, then transfected with the VEGF or GFP constructs for an additional 24 h. Right panels, quantification of protein levels from the western analysis and data are normalized to the corresponding scramble control. ***p < .001 by two-tailed unpaired t-test, n = three independent experiments. (B) Left panels, representative images of immunocytochemistry showing filopodia (white arrows) and NRP1 expression (red) in hRECs transfected with eVEGF-38 or eVEGF-53 (anti-Myc, green) 24 h after transfection with siRNA for NRP1 or a scramble control. Elongated filopodia are indicated with white arrows. Scale bar = 10 µm. Right panels, quantification of the fluorescence intensity of NRP1 immunostaining, the average length of the longest three filopodia per cell, and number of filopodia per cell. N = 15 cells from three independent experiments, ***p < .001 by two-tailed unpaired t-test, NS = not significant. All graphs show mean ± SEM

Article Snippet: Antibodies against phospho- VEGFR2 (Y1175), VEGFR2, Akt, phospho- Akt (S473), p38 MAPK, phospho- p38 MAPK, p44/42 MAPK (Erk1/2), phospho- p44/42 MAPK (Erk1/2), alpha- tubulin, and myc epitope tag were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Activation Assay, Expressing, Western Blot, Phospho-proteomics, Transfection, Control, Construct, Two Tailed Test, Immunocytochemistry, Fluorescence, Immunostaining

Journal: Journal of molecular histology

Article Title: tsRNA-3043a intensifies apoptosis and senescence of ovarian granulosa cells to drive premature ovarian failure by targeting FLT1.

doi: 10.1007/s10735-024-10256-8

Figure Lengend Snippet: Fig. 10 Diagram of the hypothesis mechanism of tsRNA-3043a facilitates POF by inhibiting FLT1

Article Snippet: The sections were blocked with 5% BSA and incubated with the anti-FLT1 antibody (1:200, 13687-1-AP, Proteintech) overnight at 4°C.

Techniques:

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: PDGFC facilitates enzalutamide resistance in prostate cancer through activation of the Rap1-MAPK pathway

doi: 10.1007/s00432-025-06276-w

Figure Lengend Snippet: PDGFC expression abnormally elevated in EnzaR PCa cells. a The mRNA expression of AR and AKR1C3 were analyzed by qRT-PCR. b The protein expression of AR and AKR1C3 were analyzed by Western blotting. c , d Cell viability was measured after the indicated cell lines were treated with different doses of enzalutamide ( c ) or at different time points ( d ). e Colony formation assay was measured in indicated EnzaR cells. The right panel shows the colony numbers. f Venn diagram showing the intersecting genes of four PCa enzalutamide resistance datasets ( GSE150807 , GSE151083 , GSE123379 , and GSE163240 ) based on the criteria of logFC >|2| and p < 0.05. g Expression of indicated genes in the patient Antonarakis ES dataset. h PDGFC expression in the corresponding GEO datasets. i The mRNA and protein expression of PDGFC in LNCaP and RM-1 cells were analyzed by qRT-PCR and Western blotting. p values are shown for each comparison (* p < 0.05; ** p < 0.01; ns, not significant)

Article Snippet: Primary antibodies were used at the following concentrations; AR (dilution 1:000, R380939, Zen-bioscience, China), AKR1C3 (dilution 1:1000, R383067, Zen-bioscience, China), PDGFC (dilution 1:1000, 55076–1-AP, Proteintech, China), RAP1A (dilution 1:1000, YN2181, immunoway, China), ERK1/2 (dilution 1:1000, 9102, CST, USA), p-ERK1/2 (dilution 1:1000, 9101, CST, USA), p38 (dilution 1:1000, 9212, CST, USA), p-p38 (dilution 1:1000, 9211, CST, USA), STAT4 (dilution 1:1000, A4523, Abclonal, China), p-STAT4 (dilution 1:1000, YP0252, immunoway, China) and β-Actin (dilution 1:1000, R380624, Zen-bioscience, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Colony Assay, Comparison

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: PDGFC facilitates enzalutamide resistance in prostate cancer through activation of the Rap1-MAPK pathway

doi: 10.1007/s00432-025-06276-w

Figure Lengend Snippet: PDGFC promotes EnzaR by activating Rap1/MAPK pathway in PCa cells. a Volcano plot showing the differentially depleted (blue) and enriched (red) genes in LNCaP-EnzaR-shPDGFC cells compared with LNCaP-EnzaR-shNC cells. b Heatmap was performed in LNCaP-EnzaR-shNC and shPDGFC. c KEGG enrichment analysis focuses on the down-regulated genes, (logFC < -2, FDR < 0.05). d The correlations between PDGFC and RAP1A, MAPK14 (P38), and MAPK3 (ERK) were analyzed through GEPIA. e The protein levels of P38, ERK1/2, p-P38, p-ERK1/2, and Rap1A were analyzed by western blotting in EnzaR cells

Article Snippet: Primary antibodies were used at the following concentrations; AR (dilution 1:000, R380939, Zen-bioscience, China), AKR1C3 (dilution 1:1000, R383067, Zen-bioscience, China), PDGFC (dilution 1:1000, 55076–1-AP, Proteintech, China), RAP1A (dilution 1:1000, YN2181, immunoway, China), ERK1/2 (dilution 1:1000, 9102, CST, USA), p-ERK1/2 (dilution 1:1000, 9101, CST, USA), p38 (dilution 1:1000, 9212, CST, USA), p-p38 (dilution 1:1000, 9211, CST, USA), STAT4 (dilution 1:1000, A4523, Abclonal, China), p-STAT4 (dilution 1:1000, YP0252, immunoway, China) and β-Actin (dilution 1:1000, R380624, Zen-bioscience, China).

Techniques: Western Blot

Journal: Respiratory research

Article Title: Altered hypoxia-induced cellular responses and inflammatory profile in lung fibroblasts from COPD patients compared to control subjects.

doi: 10.1186/s12931-024-02907-x

Figure Lengend Snippet: Fig. 1 Effect of 4 h of hypoxia exposure on gene expression levels. Genes related to remodelling: VEGFR2 (a), 5-HTR2B (b) and Collagen7 (c); cellular stress: IRE1 (d) and c-Jun (h), oxidative stress: Nrf2 (e), HIF-1α (f) and SOD3 (g) were measured by RT-qPCR in primary distal lung fibroblasts obtained from healthy subjects (n = 7) and COPD patients (n = 7) after 4 h of exposure to normoxic (21% O2) or hypoxic (1% O2) conditions. Beta-actin and 18 S were used as housekeeping genes. The data is presented as median with interquartile range. Ordinary two-way ANOVA or RM two-way ANOVA were used for unpaired and paired comparisons and the post-hoc test Fisher’s LSD was used for statistical analysis *p < 0.05, **p < 0.01

Article Snippet: The following primary antibodies were used: HIF-1α (dilution 1:50, Biotin, ab81633), HIF-2α (dilution 1:100 Novus Biologicals, Bio-Techne, #NB100132), 5HTR2B (dilution 1:300, Aviva System Biology, OAAF02801), VEGFR2 (dilution 1:200, Bio-rad, AHP1327), VEGFR3 (dilution 1:200, Abcam, GR3217142-6) and negative control (Dako, X0903), all diluted in 1% BSA in TBS.

Techniques: Gene Expression, Quantitative RT-PCR