Journal: Nature Communications
Article Title: FUNDC1-dependent mitochondria-associated endoplasmic reticulum membranes are involved in angiogenesis and neoangiogenesis
doi: 10.1038/s41467-021-22771-3
Figure Lengend Snippet: a Transduction efficiency of peptides 1, 2, and a control peptide in Human umbilical vein endothelial cells (HUVECs) ( n = 5 independent experiments). b HUVECs were treated with different peptides (20 μM) for 24 h, then subjected to immunoprecipitation with antibody against FUNDC1(HA) to quantify the interaction of FUNDC1 and IP3R1 ( n = 3 independent experiments). c HUVECs were treated with different peptides (20 μM) for 24 h, then cytosolic Ca 2+ was detected using the fluorescent probe Fluo-4, AM ( n = 5 independent experiments). d HUVECs were treated with different peptides. After 24 h, the cell lysates were collected and subjected to western blot assays to detect the expression of VEGFR2, IP3R1, SRF, and phosphorylated SRF at Ser103 (pSRF) ( n = 5 independent experiments). e Three-dimensional spheroids and representative images of spheroid-sprouting were analyzed ( n = 5 independent experiments). Scale bar, 100 µm. f Matrigel containing vascular endothelial growth factor (VEGF) was injected subcutaneously into 6-week-old wild-type mice, then the mice received an intravenous injection of the indicated peptides. After 10 days, matrigel plugs were removed for analysis of new vessel formation by histological and Hb assay ( n = 5 mice/group). Quantification of Hb extracted from matrigel plugs of different groups. g C57BL/6 mice with LLC tumors that were approximately 90 mm 3 in size were treated with intravenous injections of different peptides (10 mg/kg/3 days). After 28 days, the tumors were harvested and quantified. Representative images of tumors ( n = 5 mice/group). h Immunostaining of LLC tumor sections and quantification of relative CD31-positive area. ( n = 5 mice/group). Scale bar, 100 µm. Statistical significance was assessed using one-way ANOVA with post hoc multiple comparisons test for multiple groups. *p < 0.05 vs Ctrl peptide. All values are mean ± S.D.
Article Snippet: Antibodies against the following proteins were used as the primary antibodies: CD31 (BD Pharmingen, San Jose, CA; 550274; Cell Signaling Technology, Danvers, MA;77699); β-actin and GAPDH (Santa Cruz Biotechnology, Dallas, Texas; sc-47778 and sc-137179); PCNA, Calreticulin, Cyt C, VDAC1, mitofusin-2 (MFN2), SRF, phosphorylated (p) SRF, VEGFR1, VEGFR2, pVEGFR2, and VEGFR3 (Cell Signaling Technology, Danvers, MA; 13110, 12238, 4280,4661, 9482, 5147,4261, 2893, 2478, 2479, 3408); IP3R1 (Abcam, Cambridge, MA; ab5804; ThermoFisher, Waltham, MA, PA1-901); calnexin (Abcam, Cambridge, MA; ab22595); FUNDC1 (Aviva Systems Biology, San Diego, CA; ARP53281), FUNDC1 (Novus Biologicals, LLC, Littleton CO, NBP1-81063; EMD Millipore Corporation, Temecula, CA, ABC506).
Techniques: Transduction, Control, Immunoprecipitation, Western Blot, Expressing, Injection, Immunostaining
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![a HUVECs were pre-transfected with or without FUNDC1 siRNA for 24 h, then treated with actinomycin D for different periods of time. RT-qPCR assay analysis of vascular endothelial growth factor receptor 2 (VEGFR2) mRNA levels ( n = 5 independent experiments). b HUVECs were pre-transfected with or without FUNDC1 siRNA for 24 h, then transfected with various VEGFR2 promoter truncation constructs or a mutant (serum response factor <t>[SRF]</t> binding site) construct for 24 h. After this, luciferase activity was measured ( n = 5 independent experiments). c Chromatin immunoprecipitation assays using an anti-SRF antibody to amplify VEGFR2 promoter in HUVECs transfected with or without FUNDC1 siRNA ( n = 5 independent experiments). d HUVECs were pre-transfected with or without FUNDC1 siRNA for 24 h and then treated with VEGF for 30 min. Expression of SRF <t>and</t> <t>phosphorylated</t> SRF at Ser103 (pSRF) was determined by western blot assay ( n = 5 independent experiments). e Cytosolic Ca 2+ levels, as indicated by the fluorescent probe Fluo-4, AM ( n = 5 independent experiments). HUVECs were transfected with Scr siRNA or FUNDC1 siRNA for 24 h and then exposed to VEGF (20 ng/mL) for the indicated times. f HUVECs were infected with adenovirus encoding control (Ad-Cont) or mitochondrial–endoplasmic reticulum (ER) linker (Ad-Linker) for 24 h and then treated with BAPTA-AM (10 µM) or control dimethylsulfoxide (vehicle) for 30 min. Levels of SRF and pSRF (Ser103) were detected by western blot assay ( n = 5 independent experiments). g HUVECs were pre-transfected with or without SRF siRNA for 24 h, then infected with adenovirus encoding control or mitochondria–ER linker for 24 h. VEGFR2 protein expression was detected by western blot assay ( n = 5 independent experiments). Statistical significance was assessed using two-tailed t -tests for two groups and one-way ANOVA with post hoc multiple comparisons test for multiple groups. *p < 0.05 vs Scr siRNA; Scr siRNA+VEGF; Ad-Cont+Vehicle or Ad-Cont+Scr siRNA. All values are mean ± S.D.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0587/pmc08110587/pmc08110587__41467_2021_22771_Fig5_HTML.jpg)
