MOESM1 of A new horizon of moyamoya disease and associated health risks explored through RNF213

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Pancreatic insulin contents and morphological characteristics of AdipoR1/Akita, AdipoR1, Akita and WT mice.

<p>Pancreatic tissue samples were obtained from AdipoR1/Akita, AdipoR1, Akita and WT mice at 15–20 weeks of age. (A) Pancreatic insulin contents of AdipoR1/Akita (n = 5), Akita (n = 5), AdipoR1 (n = 5) and WT (n = 5) mice. (B) Insulin positive area per islet area in AdipoR1/Akita (n = 5), Akita (n = 5), AdipoR1 (n = 5) and WT (n = 5) mice (upper panel). Quantification was performed on more than 20 islets from each mouse. Representative images of tissues/islet cells immunostained with anti-insulin antibodies (lower panel). Scale bars indicate 50 μm. (C) Representative images of electron micrographs of islet cells in AdipoR1/Akita, AdipoR1, Akita and WT mice. Similar results were obtained from three independent experiments. Scale bars indicate 2 μm. The small boxed areas have been enlarged and are shown in the corresponding large boxes. Arrows indicate insulin secretory granules. Data are shown as mean ± SEM. There were no significant differences between AdipoR1/Akita and Akita, or between AdipoR1 and WT, whereas there was significant differences between AdipoR1/Akita and AdipoR1 (* p<0.05), and between Akita and WT (# p<0.05) by ANOVA followed by Tukey’s honestly significant difference test. NS, Not significant.</p

Growth curves and blood glucose of AdipoR1/Akita, AdipoR1, Akita and WT mice.

<p>Growth curves and blood glucose levels of AdipoR1/Akita (n = 14), Akita (n = 11), AdipoR1 (n = 12), and WT (n = 10) mice. (A) Time course of body weight changes of mice between 4 and 10 weeks of age. Blood glucose after (B) an Ad lib feed and (C) a 16 h fast in mice between 4 and 10 weeks of age. Values are expressed as mean ± SEM. There were no significant differences between AdipoR1/Akita and Akita mice, nor between AdipoR1 and WT mice, whereas there were significant differences between AdipoR1/Akita vs AdipoR1 (* p<0.05), and Akita and WT (# p<0.05) mice by ANOVA followed by Tukey’s honestly significant difference test.</p

EGFP-tagged AdipoR1 protein expression and signaling function in MIN6 cells.

<p>(A) Representative fluorescence images of MIN6 cells transfected with empty vector (control), AdipoR1 or EGFP-tagged AdipoR1 (AdipoR1-EGFP) vectors. The scale bars represent the indicated dimensions. (B) Western blot analysis of cytosolic and membrane fractions of MIN6 cells transfected with control, AdipoR1or AdipoR1-EGFP vectors using anti-AdipoR1 antibody. Expected protein sizes are AdipoR1 (43 kDa) and AdipoR1-EGFP (70 kDa). (C) Akt phosphorylation in MIN6 cells transfected with control, AdipoR1 or AdipoR1-EGFP vectors. MIN6 cells were stimulated with 5μg/ml fAd in serum-free medium after transfections. Representative Western blots for Akt (60 kDa), phospho-Akt (Ser⁴⁷³) (60 kDa) and vinculin (124 kDa), as a loading control, are shown (left panel). Quantified values represent the ratios (compared with control) of phospho-Akt/Akt (right panel). Data are represented as mean ± SEM of four independent experiments. *p < 0.05, by ANOVA followed by Tukey’s honestly significant difference test compared with control.</p

β-cell specific AdipoR1-EGFP overexpression and signaling functions in AdipoR1 mice.

<p>(A) Pancreatic tissue samples were obtained from AdipoR1 and WT mice at 8 weeks of age. Pancreatic tissue sections were double immunostained for EGFP and insulin. DNA was stained by DAPI. Representative single-channel fluorescence and merged images are shown. Red, green and blue staining correspond to signals for AdipoR1-EGFP, insulin and DNA, respectively. Similar results were obtained from three independent experiments. The scale bars indicate 25 μm. (B) RT-PCR analysis of AdipoR1-EGFP transcripts (1910 bp) in pancreata of WT, AdipoR1 and AdipoR1/Akita mice. RT-PCR products were subjected to 1% agarose gel electrophoresis. β-actin (474 bp) was used as a loading control. (C) AMPK and Akt phosphorylation in WT and AdipoR1 mice islets stimulated with or without 5 μg/ml fAd. Representative Western blots for phospho-AMPK and AMPK (62 kDa), phospho-Akt and Akt (60 kDa), and vinculin (124 kDa) as a loading control, are shown (left panel). Quantified values represent the ratios (compared with control untreated WT mice) of phospho-AMPK/AMPK and phospho-Akt/Akt (right panel). Data are presented as mean ± SEM of three independent experiments. *p < 0.05, by ANOVA followed by Tukey’s honestly significant difference test compared with control mice.</p

Serum adiponectin levels.

<p>Serum adiponectin levels of AdipoR1/Akita (n = 8), AdipoR1 (n = 5), Akita (n = 6) and WT (n = 4) mice at 15–20 weeks of age. Values are expressed as mean ± SEM. *<i>P</i> < 0.05 by ANOVA followed by Tukey’s honestly significant difference test. NS, Not significant.</p

Supplemental material for Rare variants in <i>RNF213</i>, a susceptibility gene for moyamoya disease, are found in patients with pulmonary hypertension and aggravate hypoxia-induced pulmonary hypertension in mice

<p>Supplemental material for Rare variants in <i>RNF213</i>, a susceptibility gene for moyamoya disease, are found in patients with pulmonary hypertension and aggravate hypoxia-induced pulmonary hypertension in mice by Hatasu Kobayashi, Risako Kabata, Hideyuki Kinoshita, Takaaki Morimoto, Koh Ono, Midori Takeda, Jungmi Choi, Hiroko Okuda, Wanyang Liu, Kouji H. Harada, Takeshi Kimura, Shohab Youssefian and Akio Koizumi in Pulmonary Circulation</p

Three subtypes of clinical manifestations.

<p>Three subtypes of clinical manifestations.</p

Genome-wide linkage analysis in two Japanese familial episodic pain syndrome families.

<p>Genome-wide linkage analysis was performed for eight affected and five unaffected members in Family 1, and four affected and four unaffected members in Family 2. Parametric linkage analysis was performed using 386 genetic markers (including 382 microsatellite genetic markers) that were 10 cM apart and covered 22 autosomes, as well as additional SNP markers. GeneHunter software was used.</p

Exome analysis filtering process in the three Japanese familial episodic pain syndrome families.

<p>Exome analysis was performed for three affected members in Family 1, four affected and four unaffected members in Family 2, and two affected and one unaffected member in Family 3. Exome data was processed through seven filtering steps: (1) non-synonymous, (2) read depth ≥ 8, (3) not registered in dbSNP135, (4) MAF < 0.01 in Japanese patients from 1000 Genomes database, (5) heterozygote in affected members and not present in unaffected members, (6) located on 3p22 linkage region, and (7) variants in the same gene among all three families. Numbers in boxes represent the numbers of variants after each filtering step.</p