Zebrafish <i>wnt4a</i> can activate the zebrafish <i>eaf1</i> and <i>eaf2/U19</i> promoters.

<p>(<b>A</b>) The sequence of the 1.2 kb zebrafish <i>eaf1</i> promoter and the positions of primers used for subcloning. The transcriptional initial site is indicated by +1. (<b>B</b>) Zebrafish embryos were co-injected with the <i>eaf1</i> promoter reporter and either a control vector or a vector expressing Myc-Want4a. The luciferase activity was normalized to <i>Renilla</i> and reported as the mean ± standard deviation (SD). (<b>C</b>) The sequence of the 1.0 kb zebrafish <i>eaf2/u19</i> promoter and the primers used for subcloning. The transcriptional initial site is indicated by +1. (<b>D</b>) The expression of Myc-Wnt4a was verified by Western blot using an anti-Myc antibody. (<b>E</b>) Zebrafish embryos were co-injected with the <i>eaf2/u19</i> promoter reporter and either a control vector or a vector expressing Myc-Wnt4a. The luciferase activity was normalized to <i>Renilla</i> and reported as the mean ± SD.</p

Both human EAF2/U19 and EAF1 are Wnt4 downstream factors.

<p>(<b>A</b>) The sequence of the 1.0 kb human <i>EAF1</i> promoter and the positions of primers used for subcloning. The transcriptional initial site is indicated by +1. (<b>B</b>) 293 cells were co-transfected with the <i>EAF1</i> promoter reporter and either a control vector or a vector expressing HA-Wnt4. The luciferase activity was normalized to Renilla and reported as the mean ± standard deviation (SD). (<b>C</b>) The expression of Wnt4 was verified by Western blot using an anti-HA antibody. (<b>D</b>) The partial sequence of the 3.6 kb human <i>EAF2/U19</i> promoter and the primers used for subcloning. The transcriptional initial site is indicated by +1. (<b>E</b>) .293 cells were co-transfected with the <i>EAF2/U19</i> promoter reporter and either a control vector or a vector expressing HA-Wnt4. The luciferase activity was normalized to Renilla and reported as the mean ± SD.</p

Schematic diagram of the negative feedback regulation loop between Wnt4 and the Eaf gene family.

<p>Schematic diagram of the negative feedback regulation loop between Wnt4 and the Eaf gene family.</p

Both zebrafish <i>eaf1</i> and <i>eaf2/u19</i> genes are downstream factors of zebrafish Wnt4a.

<p>(<b>A</b>) Zebrafish embryos were injected with Wnt4-MO and <i>wnt4a</i> mRNA and the levels of zebrafish <i>eaf1</i> mRNA determined by whole-mount <i>in situ</i> hybridization at 12-somite stage. (<b>B</b>) Zebrafish embryos were injected with Wnt4-MO and <i>wnt4a</i> mRNA and the levels of zebrafish <i>eaf/u19</i> mRNA determined by whole-mount <i>in situ</i> hybridization. 12s, 12 somites. (<b>C</b>) Semi-quantitative RT-PCR analysis of <i>eaf1</i> expression in zebrafish embryos (12 somites) injected with Wnt4a-MO and <i>wnt4a</i> mRNA. (<b>D</b>) Semi-quantitative RT-PCR analysis of <i>eaf2/u19</i> expression in zebrafish embryos (12 somites) injected with Wnt4a-MO and <i>wnt4a</i> mRNA.</p

Western blot analysis of human endogenous EAF1 and EAF2/U19 protein expression after the introduction of ectopic <i>Wnt4</i> expression in 293 cells.

<p>(A) Representative Western blot of EAF1 in 293 cells transfected with a control vector or a vector expressing Wnt4 using a polyclonal antibody against human EAF1. (b) EAF1 expression was normalized to β-actin. (B) Representative Western blot of EAF2/U19 in 293 cells transfected with a control vector or a vector expressing Wnt4 using a polyclonal antibody against human EAF2/U19. (b) EAF2/U19 expression normalized to β-actin.</p

Additional file 1 of Ultrasound characteristics of normal parathyroid glands and analysis of the factors affecting their display

Additional file 1

Both zebrafish <i>eaf1</i> and <i>eaf2/u19</i> suppress zebrafish <i>wnt4a</i> expression.

<p>(<b>A</b>) Whole-mount <i>in situ</i> hybridization analysis of zebrafish <i>wnt4a</i> expression in embryos injected with <i>eaf1</i> mRNA, <i>eaf2/u19</i> mRNA, Eaf1-MO and Eaf2-MO at 12-somite stage. The embryos without injection were used as control. (<b>B</b>) The expression of zebrafish <i>wnt4a</i> mRNA was suppressed by ectopic expression of zebrafish <i>eaf1</i> and <i>eaf2/u19</i> (a) and up-regulated by knockdown Eaf1 and Eaf2/U19 (b) as revealed by semi-quantitative RT-PCR analysis at 12-somte stage.</p

Both human EAF1 and EAF2/U19 bind to the human <i>Wnt4</i> promoter as revealed by chromatin immunoprecipitaion (ChIP) assays.

<p>(<b>A</b>) 293 cells were treated with formaldehyde to create cross-links between EAF1 or EAF2/U19 and chromatin. The chromatin was isolated, sheared, and immunoprecipitated (IP) using polyclonal antibodies against human EAF1 and EAF2/U19, or preimmune serum as control. The presence of chromatin fragments corresponding to the <i>Wnt4</i> gene or to the <i>β-actin</i> gene promoter was assessed by PCR using gene-specific primers. The gel shows the recovery of Wnt4 and actin gene fragments from the protein-chromatin input on the lane 3 and 6 (from left to right) as well as those recovered after immunoprecipitation with the anti-EAF1 antibody (lane 1), with the anti-EAF2/U19 antibody (lane4) and with the pre-immune serum (land 2 and 5). (<b>B</b>) Schematic diagram depicting the fragment of the EAF1, EAF2/U19 and actin genes that were amplified. The positions of PCR primers used to detect EAF1, EAF2/U19 and actin promoter fragments are indicated by arrows.</p

sj-xlsx-1-uix-10.1177_01617346241246169 – Supplemental material for Pulmonary Transit Time Assessment by CEUS in Healthy Rabbits: Feasibility, and the Effects of UCAs Dilution Concentration

Supplemental material, sj-xlsx-1-uix-10.1177_01617346241246169 for Pulmonary Transit Time Assessment by CEUS in Healthy Rabbits: Feasibility, and the Effects of UCAs Dilution Concentration by Song Kang, Jianfeng Chen, He Zhang, Guangyin Li, Yingying Liu, Xue Mei, Binyang Zhu, Xin Ai and Shuangquan Jiang in Ultrasonic Imaging</p

Representative electron micrographs of mitochondria (×100,000).

<p>Mitochondria were isolated from the swine frontal cortex at 24 h following ROSC. A: normal mitochondria with intact membrane cristae and a smooth matrix in the blank control group. B: slightly damaged mitochondria with basically normal cristae and a slightly damaged matrix in the hypothermia group. C: markedy deranged mitochondria with disrupted cristae and a damaged matrix in the normothermia group. Scale bar  = 500 µm.</p