postfertilization hpf  (Santa Cruz Biotechnology)

Journal: Leukemia

Article Title: Asxl1 C-terminal mutation perturbs neutrophil differentiation in zebrafish

doi: 10.1038/s41375-021-01121-8

Figure Lengend Snippet: A Site-specific targeting for CRISPR/Cas9 cleavage within exon 12 of the zebrafish asxl1 gene. Alignment of nucleotide sequences from wild-type and mutant asxl1 alleles in asxl1 e12 (−7) and asxl1 e12 (−22) zebrafish lines. Dashes in DNA sequences are the nucleotides deleted during repair of CRISPR/Cas9-induced double-strand breaks. PAM protospacer adjacent motif. CRISPR/cas9-induced asxl1 frameshift mutations predicted to lead to C-terminally truncated proteins. Truncated proteins predicted from mutant alleles asxl1 e12 (−7) and asxl1 e12 (−22) lack the last two domains (ASXM2 and PHD). B qRT-PCR comparing expression of asxl1 in asxl1 +/+ and mutant asxl1 −/− in 3 days postfertilization (dpf) larvae and adult kidney marrow (3 dpf, 3 dpf larvae tails, n ≥ 10 per group, performed with four replicates; 6 m, 6-month kidney marrow, n = 4 per genotype; two-tailed Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001; error bars, mean ± standard deviation (SD)). C Gross appearance of asxl1 −/− zebrafish compared with asxl1 +/+ littermates (at 1.5 years). Body weights of asxl1 +/+ and asxl1 −/ − zebrafish (scale bar, 1 cm; asxl1 +/+ , n = 33; asxl1 −/− , n = 24; two-tailed Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001, error bars, mean ± SD).

Article Snippet: Embryos at 24 h postfertilization (hpf) were soaked in egg water containing UNC3866 (Selleckchem, S8359), PRT4165 (Selleckchem, S5315), CPI455 HCl (Selleckchem, S8287), or GSK J4 (Selleckchem, S7581).

Techniques: CRISPR, Mutagenesis, Quantitative RT-PCR, Expressing, Two Tailed Test, Standard Deviation

Journal: Journal of the American Heart Association

Article Title: Role of ADTRP (Androgen‐Dependent Tissue Factor Pathway Inhibitor Regulating Protein) in Vascular Development and Function

doi: 10.1161/jaha.118.010690

Figure Lengend Snippet: Figure 1. Expression of adtrp in zebrafish embryos. A through E, Whole-mount in situ hybridization for adtrp in zebrafish embryos using digoxigenin-labeled riboprobes. A, Adtrp expression in anterior, medial, and posterior lateral plate mesoderm (lpm; arrows). B through D, Expression of adtrp is apparent in major blood vessels (arrows) at 16, 24, and 48 hours postfertilization (hpf). C, Right panel displays a cross-section of 24-hpf embryo confirming adtrp expression in dorsal aorta and cardinal vein. Bar: 50 lm. E, Negative control with sense riboprobe. F, mRNA expression of adtrp assayed by qPCR in zebrafish embryos through the embryonic development. n>30 biological replicates for each time point. cv indicates cardinal vein; da, dorsal aorta; nt, notochord.

Article Snippet: For blocking Mmp-9 activity, dechorionated embryos at 48 hours postfertilization (hpf) were incubated with 100 lmol/L of GM600124–27 (Santa Cruz Biotechnology) and harvested at 72 hpf.

Techniques: Expressing, In Situ Hybridization, Labeling, Negative Control

Journal: Journal of the American Heart Association

Article Title: Role of ADTRP (Androgen‐Dependent Tissue Factor Pathway Inhibitor Regulating Protein) in Vascular Development and Function

doi: 10.1161/jaha.118.010690

Figure Lengend Snippet: Figure 2. Efficiency of adtrp knockdown in zebrafish embryos. A, Design of splice-blocking morpholinos (MO) for adtrp1, the adtrp homologous gene on chromosome (Chr) 23; hereafter named “adtrp,” targeting exon2-intron2 (e2i2) and exon3-intron3 (e3i3) boundaries. B, mRNA expression of adtrp assayed by qPCR in 72 hours postfertilization (hpf) embryos shows 50% knockdown efficiency for both adtrp MOs used at 1 pmol MO/embryo. adtrpe2i2 MO, hereafter named “adtrp” MO, was used in all knockdown experiments unless otherwise stated. Statistics: 1-way ANOVA with Bonferroni’s multi- comparison test. Values are meanSEM fold-change from control after correction for elongation factor 1a as internal control. ****P<0.0001. C, Effect of adtrp1 MO injection on mRNA expression of adtrp2 (adtrp homologous gene on Chr 13), assayed by qPCR in 72-hpf embryos. Statistics: unpaired t test. Data are presented as for (B). NS indicates not significant, P>0.05. B and C, Experiments were repeated 3 times with qPCR technical duplicates. Each marker represents a group of at least 10 embryos.

Article Snippet: For blocking Mmp-9 activity, dechorionated embryos at 48 hours postfertilization (hpf) were incubated with 100 lmol/L of GM600124–27 (Santa Cruz Biotechnology) and harvested at 72 hpf.

Techniques: Knockdown, Blocking Assay, Expressing, Comparison, Control, Injection, Marker

Journal: Journal of the American Heart Association

Article Title: Role of ADTRP (Androgen‐Dependent Tissue Factor Pathway Inhibitor Regulating Protein) in Vascular Development and Function

doi: 10.1161/jaha.118.010690

Figure Lengend Snippet: Figure 3. Knockdown of adtrp causes vascular defects in zebrafish embryos. A, Bright-field (left panels) and whole-mount fluorescence confocal microscopy of 72 hours postfertilization (hpf) Tg(fli1:EGFP)y1 zebrafish embryos (right panels; EGFP, fluorescent vascular reporter) injected with scrambled (control) or adtrp morpholinos (MO)human ADTRP mRNA. Human FLAG-tagged ADTRP expression was verified by western blot (lower panel blot of 72-hpf embryo lysates; protein loading: Ponceau Red staining). White arrows: intersegmental vessels (ISVs). White dashed line: caudal vein plexus (CVP) area. Bars: 100 lm. Quantification of vascular phenotype: scatter plots and statistics (1- way ANOVA with Bonferroni’s multicomparison test) of B the percentage of defective ISVs, and C the percentage area of CVP dilation normalized to tail area. Data are meanSEM of biological replicates (n). ****P<0.0001 between all groups. D, Semithin sections at the CVP level (red dashed lines) of control (a) and adtrp MOs (b and c). White arrow: endothelial cells (ECs). Bars: a and b, 10 lm; c, 5 lm. E, O-dianisidine staining for red blood cells in 72-hpf embryos injected with adtrp or control MOs. Arrows: hemorrhage spots. F, Fluorescence confocal microscopy of 72-hpf Tg(fli1-EGFP)y1 embryos injected with adtrpe2i2+p53, adtrpe3i3, or p53 MOs shows nearly identical defective patterning of ISV (arrows) and dilated CVP (white dashed line areas) in all adtrp morphants, but not in the p53 MO– injected embryos. Bars: 100 lm. ADTRP indicates androgen-dependent tissue factor pathway inhibitor regulating protein; EGFP, enhanced green fluorescent protein.

Article Snippet: For blocking Mmp-9 activity, dechorionated embryos at 48 hours postfertilization (hpf) were incubated with 100 lmol/L of GM600124–27 (Santa Cruz Biotechnology) and harvested at 72 hpf.

Techniques: Knockdown, Confocal Microscopy, Injection, Control, Expressing, Western Blot, Staining, Fluorescence

Journal: Journal of the American Heart Association

Article Title: Role of ADTRP (Androgen‐Dependent Tissue Factor Pathway Inhibitor Regulating Protein) in Vascular Development and Function

doi: 10.1161/jaha.118.010690

Figure Lengend Snippet: Figure 16. Adtrp deficiency leads to increased expression of matrix metallopeptidase-9 (Mmp9) mRNA, protein, and activity levels in zebrafish embryos and newborn mice. A, Whole- mount in situ hybridization for mmp9 (blue) in 72 hours postfertilization (hpf) control and adtrp zebrafish morphants. Blue arrow: specific signal accumulation. Black arrow: caudal vascular plexus (CVP). n, biological replicates. Bars, 100 lm. B, Immunofluorescence staining with anti- Mmp-9 (Cy3, red) IgG on cross-sectioned CVP (green) of 72-hpf Tg(fli1:EGFP)y1 (EGFP, fluorescent vascular reporter) control and adtrp morphantshuman ADTRP mRNA. Bars, 100 lm. C, Total lysates of 72-hpf zebrafish embryos incubated 100 lmol/L of pan-MMP inhibitor GM6001 for 24 hours and analyzed by 10% gelatin gel electrophoresis. Left: representative zymogram (grayscale inverted image). Histogram: semiquantitative densitom- etryandstatistics(meanSEMbyttest).*P<0.05.Eachmarkerrepresentsagroupofatleast10 embryos. D, In situ zymography with MMP fluorescence substrate QXL TM 570-KPLA-Nva-Dap(5- TAMRA)-AR-NH2 (red, dequenched fluorescence) on cross-sectioned CVP (green) of 48-hpf Tg (fli1:EGFP)y1 embryos injected with Control or adtrp morpholinos (MOs). Arrows: MMP activity. E,Fluorescenceconfocalmicroscopyof72-hpfTg(fli1:EGFP)y1embryosinjectedwithcontrolor adtrp MOs (left panels; EGFP, fluorescent vascular reporter)GM6001 as for C. Bars, 100 lm. Quantificationof vascular defects (right panels):scatter plots and statistics (1-way ANOVA with Bonferroni’smulticomparisontest)ofthepercentageofdefectiveintersegmentalvessels(ISVs) and percentage area of CVP dilation normalized to tail area. Data are meanSEM of biological replicates (n). ****P<0.0001 between all groups. F, Double immunofluorescence and whole- mountconfocalmicroscopywithanti-MMP-9(Cy3,red)andeitheranti-CD31oranti-GR-1(FITC, green) in cephalic skin; or with anti-MMP-9 (Cy3, red) and anti-mast cell (MC) Tryptase (FITC, green) in lung of wild-type (WT) and Adtrp/ P0 pups. MMP-9 associates with both CD31 and GR-1–positive cells in the skin (lower panels, white arrow and inset), and with MC (lung panels, yellow arrows). Bars, 100 lm. G, Zymography (as in C) of conditioned media of primary fibroblasts cultured from P0 WT and Adtrp/ cephalic skin explants. Upper panel: representative zymogram (grayscale inverted image). Histogram: semiquantitative densitom- etry and statistics using t test. Data are meanSEM of biological replicates. ****P<0.0001. Adtrp indicates androgen-dependent tissue factor pathway inhibitor regulating protein; EGFP, enhanced green fluorescent protein; IgG indicates immunoglobulin G.

Article Snippet: For blocking Mmp-9 activity, dechorionated embryos at 48 hours postfertilization (hpf) were incubated with 100 lmol/L of GM600124–27 (Santa Cruz Biotechnology) and harvested at 72 hpf.

Techniques: Expressing, Activity Assay, In Situ Hybridization, Control, Staining, Incubation, Nucleic Acid Electrophoresis, In Situ, Zymography, Injection, Cell Culture

Journal: Journal of the American Heart Association

Article Title: Role of ADTRP (Androgen‐Dependent Tissue Factor Pathway Inhibitor Regulating Protein) in Vascular Development and Function

doi: 10.1161/jaha.118.010690

Figure Lengend Snippet: Figure 17. ADTRP deficiency-induced matrix metallopeptidase- 9 (MMP-9) is regulated by canonical Wnt signaling. A, Double immunofluorescence with anti-MMP-9 (FITC, green) and anti-b- Galactosidase (b-GAL; Cy3, red), and confocal microscopy in whole-mount P0 pups lung of Adtrp/BAT-GALz+ and wild-type (WT) BAT-GALz+. Arrows: partial expression of b-GAL (active b- catenin reporter) in MMP-9–producing cells. Blue, nuclei. Bars, 50 lm. B, mRNA expression of Mmp9 in primary fibroblasts cultured from WT and Adtrp/ P0 pups cephalic skin explants. Gray bars: treatment with Wnt3a medium for 24 hours. Values are fold-change from WT (arbitrarily set to 1.0) after correction for b-2 Microglobulin as internal control. Data are meanSEM by 1- way ANOVA with Bonferroni’s multicomparison test. Markers represent mean values of 3 replicate cell cultures isolated from 2 mice each. **P<0.01; ***P<0.001. C, mRNA expression of MMP9 in control and siADTRP-EA.hy926 ECs incubated with Wnt3a medium for 24 hours. Markers represent different experiments and are fold-change from siControl (arbitrarily set to 1.0) after correction for b-2 MICROGLOBULIN as internal control. Data are meanSEM by unpaired t test. ****P<0.0001. D, mRNA expression of mmp9 in control and adtrp morphants at 72 hours postfertilization after incubation with Wnt inhibitor IWR-1 (20 lmol/L) for 8 hours. Values are fold-change from control (arbitrarily set to 1.0) after correction for b-actin or elongation factor 1a as internal controls. Each marker represents a group of at least 10 embryos. Data are meanSEM by 1-way ANOVA with Bonferroni’s multicomparison test. **P<0.01; ****P<0.0001. ADTRP indicates androgen-dependent tissue factor pathway inhibitor regulating protein; ECs, endothelial cells.

Article Snippet: For blocking Mmp-9 activity, dechorionated embryos at 48 hours postfertilization (hpf) were incubated with 100 lmol/L of GM600124–27 (Santa Cruz Biotechnology) and harvested at 72 hpf.

Techniques: Confocal Microscopy, Expressing, Cell Culture, Control, Isolation, Incubation, Marker