Journal: Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology

Article Title: Regulation of vascular signalling by nuclear Sprouty2 in fetal lung epithelial cells: Implications for co-ordinated airway and vascular branching in lung development

doi: 10.1016/j.cbpb.2018.01.007

Figure Lengend Snippet: Nuclear expression of Spry2 in FDLE. A. Immunofluorescence shows Spry2 is nuclear in rat FDLE cells and this distribution is conserved irrespective of culture PO 2 or FGF-10. Image is representative of 4 independent preparations m – membrane, n – nucleus. Neg control, no primary antibody B. Spry2 resolves as 39 and 35 kDa in rat FDLE nuclear lysates; H3, Histone 3 (i). FGF-10 induces clearance 39 kDa Spry2, (ii) Spry2 siRNA (rat FDLE, 250 pmol·ml −1 ) and stable shRNA (HBE) suppresses 35 and 39 kDa Spry2 abundance. (iii) Quantitation of FGF-10 effect upon 39 kDa 35 kDa Spry2 in FDLE, mean ± SEM, n = 6, *P < 0.05 relative to control. (iv) The effect of FGF-10 upon 39 kDa Spry clearance is not altered by culture PO 2 does not alter irrespective of culture PO 2 , mean ± SEM, n = 6. C. Western blotting of cytosolic and nuclear Spry2 in 16HBE14o- (HBE) cells stably expressing dual tagged Spry2 (upper image). LiCor analysis simultaneously resolved N-terminal FLAG (red) and C-terminal Myc (green) epitope tags in cytosolic, nuclear-soluble and nuclear-insoluble fractions from cells treated for up to 4 h in 0.1 μg·ml −1 FGF-10. Yellow in the merged image indicates co-localisation of the tags. The nuclear scaffold protein, p84, served as a loading control. Blots representative of 4 independent experiments. D. Immunofluorescence shows native (i–iii) and dual tagged expressed Spry2 (iv–vi) occurs in the nucleus in HBE cells and associates with the spindle pole in mitotic cells (right). Images representative of 4 independent experiments; Abbreviations: m, membrane; n, nucleus; s, mitotic spindles; Ac Tub, Acetylated α-tubulin. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Non-genome target control (TR20003) and shRNA vectors targeting human Spry2 in pRS were originally purchased from Origene Technologies, Inc. (Rockville, MD; Catalog number TR309130) and kindly supplied to us by Drs P Yusoff and G Guy (Proteos, Singapore).

Techniques: Expressing, Immunofluorescence, shRNA, Quantitation Assay, Western Blot, Stable Transfection

Journal: Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology

Article Title: Regulation of vascular signalling by nuclear Sprouty2 in fetal lung epithelial cells: Implications for co-ordinated airway and vascular branching in lung development

doi: 10.1016/j.cbpb.2018.01.007

Figure Lengend Snippet: FGF-10 induces histone modification in FDLE and correlates with the decline in 39 kDa Spry2. A. FGF-10-evoked decline in 39 kDa Spry2 in nuclear and cytosolic fractions corresponds with increased Histone 3 S10(P)K14(Ac) phosphoacetylation and decreased nuclear abundance of HDAC1-3. B. (i) Relationship between H3 S10(P)K14(Ac) phosphoacetylation and FGF-10 concentration and (ii) 39 kDa Spry2 abundance (FGF-10 concentration (μg·ml −1 ) shown next to symbols). Values are Mean ± SEM, n = 4; *P < 0.01 relative to control.

Article Snippet: Non-genome target control (TR20003) and shRNA vectors targeting human Spry2 in pRS were originally purchased from Origene Technologies, Inc. (Rockville, MD; Catalog number TR309130) and kindly supplied to us by Drs P Yusoff and G Guy (Proteos, Singapore).

Techniques: Modification, Concentration Assay

Journal: Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology

Article Title: Regulation of vascular signalling by nuclear Sprouty2 in fetal lung epithelial cells: Implications for co-ordinated airway and vascular branching in lung development

doi: 10.1016/j.cbpb.2018.01.007

Figure Lengend Snippet: FGF-10 does not alter HIFα subunit stability but increases VEGF-A expression and protein recruitment to the VEGF-A HRE in FDLE. A. Western blot of nuclear HIFα proteins, Spry2 and Histone 3 (H3) from FDLE exposed to increasing concentrations of FGF-10. CoCl 2 (100 μM, 16 h), positive control, representative of 4 independent experiments. B. mRNA and protein abundance of VEGF-A exposed to FGF-10 measured by qPCR and ELISA, mean ± SEM, n = 4, *P < 0.05. C. EMSA showing retarded migration of [ 32 P]-VEGF-A HRE probe incubated with 10 μg of nuclear lysate from FDLE cells treated with FGF-10 as indicated. Mutant probe (Mut) shows non-specific binding; Free probe, unincorporated excess probe; dotted line shows position of band at 0 μg·ml −1 .FGF-10 D. Supershift EMSA showing decreased mobility of bands incubated with antibodies (1 μg) against Spry2 and HIF-1-3α. Non-immune IgG, control. E. qPCR analysis showing effect of stable Spry2 knockdown in HBE cells (Spry2 shRNA) maintained at fetal PO 2 upon transcript abundance of Spry2, HIF-1α VEGF-A (regulated by HIF-1α and HIF-2α) BNIP3 (regulated by HIF-1α) and CCND1 (regulated by HIF-2α). Values are mean ± SEM, n = 4, *P < 0.05.

Article Snippet: Non-genome target control (TR20003) and shRNA vectors targeting human Spry2 in pRS were originally purchased from Origene Technologies, Inc. (Rockville, MD; Catalog number TR309130) and kindly supplied to us by Drs P Yusoff and G Guy (Proteos, Singapore).

Techniques: Expressing, Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay, Migration, Incubation, Mutagenesis, Binding Assay, shRNA

Journal: Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology

Article Title: Regulation of vascular signalling by nuclear Sprouty2 in fetal lung epithelial cells: Implications for co-ordinated airway and vascular branching in lung development

doi: 10.1016/j.cbpb.2018.01.007

Figure Lengend Snippet: Nuclear Spry2 interacts with 5′ elements in the rat VEGF-A promoter. FDLE cells were incubated at fetal PO 2 in the absence or presence of FGF-10 at the given concentrations. ChIP was performed using primers designed to amplify 5′ promoter elements as indicated in the diagram (right). Sequence alignments are to scale and are for the rat VEGF-A promoter; the position of the HIF consensus site is shown. Arrow at 3′ end indicates position of classical transcriptional initiation site (TIS). Result is representative of at least 4 independent experiments.

Article Snippet: Non-genome target control (TR20003) and shRNA vectors targeting human Spry2 in pRS were originally purchased from Origene Technologies, Inc. (Rockville, MD; Catalog number TR309130) and kindly supplied to us by Drs P Yusoff and G Guy (Proteos, Singapore).

Techniques: Incubation, Sequencing

Journal: Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology

Article Title: Regulation of vascular signalling by nuclear Sprouty2 in fetal lung epithelial cells: Implications for co-ordinated airway and vascular branching in lung development

doi: 10.1016/j.cbpb.2018.01.007

Figure Lengend Snippet: A region within the Spry2 cysteine rich domain (CRD) is necessary for Spry2 to interact with a region spanning the human VEGF-A HIF consensus site. A(i.) Sequence alignment of amino acids 199–221 in human Spry2 with Spry1, 3 and 4 with residues in Spry2 showing homology with the RanBP2 Zn 2+ finger signature given in bold and underlined. (ii) Species conservation of the Spry2 sequence given in (i). B. ChIP assay against a region of the human VEGF-A promoter spanning the HIF consensus site. Stable HBE cell lines were generated that expressed either empty vector, wild-type Spry2 (Spry2-WT), Spry2-C218A or Spry2-C221A. ChIPs performed with Anti-Spry2 or Anti-Myc (C-terminal epitope) were performed in independent samples (2 each). C. HIF-1α and Spry2 nuclear abundance are unaffected but Histone-3 S10K14 phosphoacetylation is enhanced in stable HBE cells expressing C218A or C221A Spry2 mutants. *P < 0.05 relative to Spry2-WT, n = 5 D. qPCR showing augmented VEGF-A mRNA expression in HBE cells bearing Spry2-C218A or Spry2-C221A mutants. Values are mean ± SEM, n = 4, *P < 0.05 relative to Spry2-WT.

Article Snippet: Non-genome target control (TR20003) and shRNA vectors targeting human Spry2 in pRS were originally purchased from Origene Technologies, Inc. (Rockville, MD; Catalog number TR309130) and kindly supplied to us by Drs P Yusoff and G Guy (Proteos, Singapore).

Techniques: Sequencing, Generated, Plasmid Preparation, Expressing

Journal: Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology

Article Title: Regulation of vascular signalling by nuclear Sprouty2 in fetal lung epithelial cells: Implications for co-ordinated airway and vascular branching in lung development

doi: 10.1016/j.cbpb.2018.01.007

Figure Lengend Snippet: Manipulation of Spry2 function by FGF-10, shRNA knockdown or CRD mutation promotes de-methylation of the regions flanking the VEGF-A TIS in HBE cells. A. Sequence of the region flanking the human VEGF-A TIS (grey). Putative CpG methylation sites are highlighted (underlined) B. High resolution melt (HRM) analysis of VEGF-A promoter methylation. Control melt curves for 5′ and 3′ VEGF-A flanking sequences are shown at the top and were generated using 100% methylated (solid line) or 100% non-methylated DNA (dashed line). Corresponding melt curves for FGF-10 treated samples and control versus Spry2 shRNA are shown beneath respectively. Solid line: primers against methylated DNA; Dashed line: primers against unmethylated DNA. Leftward shift in HRM curve (arrows) indicates lowered incidence of CpG methylation. Peaks in heteroduplex region suggest non-uniform CpG methylation between VEGF-A alleles. C. As for panel A but relationship is shown for HBE cells stably expressing control (wild-type) Spry2, Spry2 C218A and Spry2 C221A. Graphs are representative of 3 independent experiments.

Article Snippet: Non-genome target control (TR20003) and shRNA vectors targeting human Spry2 in pRS were originally purchased from Origene Technologies, Inc. (Rockville, MD; Catalog number TR309130) and kindly supplied to us by Drs P Yusoff and G Guy (Proteos, Singapore).

Techniques: shRNA, Mutagenesis, Methylation, Sequencing, CpG Methylation Assay, Generated, Stable Transfection, Expressing

Journal: Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology

Article Title: Regulation of vascular signalling by nuclear Sprouty2 in fetal lung epithelial cells: Implications for co-ordinated airway and vascular branching in lung development

doi: 10.1016/j.cbpb.2018.01.007

Figure Lengend Snippet: Proposed model of Spry2-regulated VEGF-A gene expression. A. Hypoxic induction of HIF-1α is held in check by interaction of high and low molecular weight Spry2 isoforms with 5′ regions of the VEGF-A promoter. Under these conditions HIF transcriptional activation is rendered inefficient due to weak binding to CBP/P300 ( ; ), CpG island methylation, histone deacetylation and DNA condensation (this study). B. FGF-10 signalling through its receptor, FGFR2b, induces Spry2- and Cbl-dependent clearance of the mTOR repressor complex, TSC1/2. The active mTOR complex 1 (mTORC1) binds to an mTOR binding motif (TOS) on HIF-1α and increases the efficiency of HIF-driven gene expression by promoting its interaction with p300/CBP ( ; ; ; ). VEGF-A promoter activity is raised by clearance of Spry2 from non GC-rich regions including the HRE; Spry2 that remains bound to GC rich regions will be in the 35 kDa form. Subsequent histone phospho-acetylation and chromatin de-condensation augments VEGF-A gene expression in proportion to growth factor signal strength.

Article Snippet: Non-genome target control (TR20003) and shRNA vectors targeting human Spry2 in pRS were originally purchased from Origene Technologies, Inc. (Rockville, MD; Catalog number TR309130) and kindly supplied to us by Drs P Yusoff and G Guy (Proteos, Singapore).

Techniques: Expressing, Molecular Weight, Activation Assay, Binding Assay, Methylation, Activity Assay