Additional file 1: Figure S1. of Characterisation of neurons derived from a cortical human neural stem cell line CTX0E16

Expression profile of undifferentiated and differentiated (DD28) CTX0E16 cells. Undifferentiated CTX0E16 cells express a range of neurotransmitter receptors, signalling and disease-associated proteins, which are upregulated during differentiation, as determined by RT-PCR; n = 3 independent experiments carried out in triplicate. Table S1 List of antibodies used in study. Table S2 List and description of primers used in RT-PCR and q-PCR experiments. (PDF 181 kb

Carex shimidzensis Franchet

原著和名: アヅマナルコ科名: カヤツリグサ科 = Cyperaceae採集地: 新潟県 岩船郡 坂町郊外 (越後 岩船郡 坂町郊外)採集日: 1972/5/19採集者: 萩庭丈壽整理番号: JH005648国立科学博物館整理番号: TNS-VS-955648備考: DB作成協力会による補足あ

Additional file 5: Figure S3. of The ACVR1 R206H mutation found in fibrodysplasia ossificans progressiva increases human induced pluripotent stem cell-derived endothelial cell formation and collagen production through BMP-mediated SMAD1/5/8 signaling

WT and FOP iECs show similar qualitative migration properties to VEGF in a transwell assay. Scale bars, 100 Οm. (TIF 1177 kb

Additional file 6: Figure S4. of The ACVR1 R206H mutation found in fibrodysplasia ossificans progressiva increases human induced pluripotent stem cell-derived endothelial cell formation and collagen production through BMP-mediated SMAD1/5/8 signaling

Characterization of osteogenic and chondrogenic potential of FOP iECs. Gene expression analysis of SMA, SOX9, MMP10, SP7, and RUNX2 by qPCR of WT and FOP iECs. None of these gene expression levels were statistically different by Student’s t test. Error bars represent mean ± one standard deviation of at least three independent replicates for each of the three WT and four FOP iEC lines. (TIF 158 kb

Six-Lead ECG analysis of AKAP13-ΔGEF mutant mice.

<p>Heart rate is in beats per minute, ms = milliseconds.</p><p>Values are given as the mean ± standard deviation for six mice in each genotype.</p

AKAP13 Rho-GEF and PKD-Binding Domain Deficient Mice Develop Normally but Have an Abnormal Response to β-Adrenergic-Induced Cardiac Hypertrophy

<div><p>Background</p><p>A-kinase anchoring proteins (AKAPs) are scaffolding molecules that coordinate and integrate G-protein signaling events to regulate development, physiology, and disease. One family member, AKAP13, encodes for multiple protein isoforms that contain binding sites for protein kinase A (PKA) and D (PKD) and an active Rho-guanine nucleotide exchange factor (Rho-GEF) domain. In mice, AKAP13 is required for development as null embryos die by embryonic day 10.5 with cardiovascular phenotypes. Additionally, the AKAP13 Rho-GEF and PKD-binding domains mediate cardiomyocyte hypertrophy in cell culture. However, the requirements for the Rho-GEF and PKD-binding domains during development and cardiac hypertrophy are unknown.</p><p>Methodology/Principal Findings</p><p>To determine if these AKAP13 protein domains are required for development, we used gene-trap events to create mutant mice that lacked the Rho-GEF and/or the protein kinase D-binding domains. Surprisingly, heterozygous matings produced mutant mice at Mendelian ratios that had normal viability and fertility. The adult mutant mice also had normal cardiac structure and electrocardiograms. To determine the role of these domains during β-adrenergic-induced cardiac hypertrophy, we stressed the mice with isoproterenol. We found that heart size was increased similarly in mice lacking the Rho-GEF and PKD-binding domains and wild-type controls. However, the mutant hearts had abnormal cardiac contractility as measured by fractional shortening and ejection fraction.</p><p>Conclusions</p><p>These results indicate that the Rho-GEF and PKD-binding domains of AKAP13 are not required for mouse development, normal cardiac architecture, or β-adrenergic-induced cardiac hypertrophic remodeling. However, these domains regulate aspects of β-adrenergic-induced cardiac hypertrophy.</p></div

Full-length AKAP13 mRNA levels are reduced by the gene-trap events.

<p>(A) TaqMan gene expression assays were used to measure the expression of AKAP13 transcripts at the indicated exon-exon junctions (E4-5, Brx-9, & E37-38). (B) Quantitative PCR analysis of wild-type (WT), heterozygote (Het) and homozygote (Hom) neonatal mouse heart and lung RNA for AKAP13 showed that none of the gene-trap mutations affected expression of the E4-5 exon-exon junction. The ΔBrx gene-trap dose dependently decreased expression of the Brx-9 exon-exon junction. Expression of the Brx-9 junction was eliminated in the AKAP13^ΔBrx/ΔBrx mice. All three gene-traps decreased expression of the E37-38 exon-exon junction in a dose-dependent manner. Expression of the E37-38 junction was eliminated in the AKAP13^ΔGEF/ΔGEF mice. The means and standard deviations are graphed for six mice per genotype. One-way ANOVA and Bonferroni’s multiple comparison tests were conducted (Prism 5; GraphPad). †, p<0.10; *, p<0.05; **, p<0.01.</p

AKAP13 is expressed in adult heart, kidney, and brain.

<p>Adult AKAP13^+/ΔGEF organs were bisected and stained with X-Gal (in blue) to determine AKAP13-βGeo expression in heart (A), kidney (C) and brain (D). (A) The AKAP13-ΔGEF hearts showed strong staining throughout the entire heart, including the left (la) and right (ra) atria, left (lv) and right (rv) ventricles, pulmonary artery, and aorta. (B) AKAP13-ΔPKD hearts had staining in the atria pulmonary artery, and aorta, as expected, but lacked staining in the ventricles. The blood vessels of the ventricles stained positive. (C) The kidney cortex (c), ureter (u), and arteries (ar) stained positive. (D) The interior of the right hemisphere of the brain showed staining of the olfactory bulb (ob), vasculature (arrow), and part of the cerebellum (cbx). Black scale bars are 1 mm.</p

The gene-trap induced truncations of AKAP13 disrupt the expected protein domains.

<p>(A) Expression constructs corresponding to the AKAP13 gene-trap events were generated using V5-tagged AKAP-Lbc truncation mutants. (B-D) These expression constructs were transfected into HEK293 cells and protein complexes were co-immunoprecipitated using anti-V5 antibody. (B) Rho-GEF activity was measured after immunoprecipitation (IP). Both AKAP-Lbc-ΔGEF and -ΔBrx had disrupted Rho-GEF activity, compared to AKAP-Lbc-WT and -ΔPKD. Immunoblotting (IB) for AKAP-Lbc-V5 with anti-V5 antibody confirmed that the AKAP-Lbc truncation mutants were expressed and immunoprecipitated at an equivalent extent. (C) Protein kinase D (PKD) activity was measured following IP. The AKAP-Lbc-ΔPKD, -ΔGEF, and -ΔBrx protein complexes lacked PKD activity compared to AKAP-Lbc-WT. Immunoblotting for GFP-PKD1 with anti-GFP antibody confirmed that only AKAP-Lbc-WT bound PKD1. The bottom gel image confirmed that GFP-PKD1 was expressed at the same level in all conditions. (D) Protein kinase A (PKA) activity was measured after IP. All AKAP-Lbc truncation mutants immunoprecipitated PKA activity and bound PKAc. The means and standard deviations are graphed for three independent experiments. One-way ANOVA and Bonferroni’s multiple comparison tests were conducted (Prism 5; GraphPad). *, p<0.05; ***, p<0.001.</p

AKAP13-ΔGEF mutant mice had normal cardiac structure.

<p>(A) Hearts isolated from six wild-type (WT) and six AKAP13^ΔGEF/ΔGEF (ΔGEF) adult male mice at 16–18 weeks of age had normal gross morphology; representative images shown. White scale bar is 1 mm. (B) WT and ΔGEF hearts were the same size as measured by heart weight to tibia length (HW/TL) ratios (in milligrams per millimeter). Means and standard deviations are graphed for six hearts of each genotype. Hearts were sectioned for histology and stained with (C) H&E or (D) Masson’s trichrome. The bottom panels of C&D are higher magnifications of the boxed regions in the top panels. (C) Cardiac structure was normal in ΔGEF hearts (top), and cardiomyocytes had proper organization (bottom). (D) ΔGEF hearts had normal levels of fibrosis as assessed by Masson’s trichrome staining. Black scale bars in C&D are 1 mm (top), 50 µm (C bottom), and 250 µm (D bottom).</p