Journal: Stem Cell Research & Therapy

Article Title: Growth differentiation factor 11 alleviates oxidative stress-induced senescence of endothelial progenitor cells via activating autophagy

doi: 10.1186/s13287-024-03975-y

Figure Lengend Snippet: Rat bone marrow-derived CD34 + VEGFR-2 + EPCs. ( A ) Analysis of CD34 + VEGFR-2 + EPCs by flow cytometry. ( B ) CD34 + VEGFR-2 + EPCs (arrows) in the mononuclear cells isolated from bone marrow. Immunostaining. Scale bar = 25 μm. ( C ) CD31 expression in CD34 + VEGFR-2 + EPCs at 2 weeks after induction with VEGF. Immunostaining. Scale bar = 10 μm. ( D , E ) ALK-4 expression in EPCs demonstrated with immunostaining and Western blotting respectively. Scale bar = 25 μm. ( F ) The statistical result of ALK-4 expression. * P < 0.01 versus young group. n = 3

Article Snippet: The cells were then incubated with mouse anti-rat CD34 and rabbit anti-rat VEGFR-2 antibodies (1:100; Santa Cruz Biotech, Dallas, TX, USA) at 4 o C for 50 min, followed by incubation with Alexa Fluor 647-conjugated goat anti-mouse IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; Jackson, West Grove, PA, USA) at 4 o C for 30 min. CD34 + VEGFR-2 + cells in the mononuclear cells were sorted using a Beckman MoFloTM XDP FACS (fluorescence-activated cell sorter; Beckman Coulter, Fullerton, CA, USA).

Techniques: Derivative Assay, Flow Cytometry, Isolation, Immunostaining, Expressing, Western Blot

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