Journal: Nucleic Acids Research

Article Title: Ecd promotes U5 snRNP maturation and Prp8 stability

doi: 10.1093/nar/gkaa1274

Figure Lengend Snippet: Ecd Δ34 mutant protein binds R2TP complex via PIH1D1 but is non-functional in vivo . ( A ) Schematic representation of wild-type and mutant Ecd proteins and ecd alleles used in this study. The blue boxes indicate the position of wild-type (dark blue) or mutated (light blue) DSDD/DEDD motifs. Alignment of the C-terminal part of the Drosophila melanogaster ( D.m ., Q9W032) Ecd and human ( H.s. , O95905) ECD proteins was generated using Clustal W (A’). Asterisks indicate the positions of the premature stop codon in ecd l( 3 )23 allele and the conserved proline 656, which substitution to serine generates conditional ecd 1 allele. Black and red rectangles outline the DSDD and DEDD motifs, respectively (A’). ( B , C ) Representative western blots and quantifications show that Myc::Ecd TripleA (B) but not Myc::Ecd Δ34 (C) co-precipitates significantly less Flag::PIH1D1 protein from Drosophila S2 cell lysates relative to Myc::Ecd wt (B, C). Myc-tagged proteins served as baits. GFP was used as a transfection and loading control. Data represent means ± SD, n = 3. Unpaired two-tailed Student's t -test was used to determine the significance, *** P < 0.001, n.s. = non-significant. ( D–M ) Representative confocal micrographs of mosaic third instar EADs and brightfield and fluorescent images of adult eyes, where homozygous GFP-labelled clones of the indicated genotypes were generated using the eyFLP MARCM technique. In contrast to abundant, sizable control clones (D, E), ecd Δ homozygous mutant clones are very rare, presented as individual GFP-positive cells within the differentiated part of the eye primordium (F) and adult retina (G). Overexpression of Ecd wt (H, I) and Ecd TripleA (J, K) but not Ecd Δ34 mutant protein (L, M) is sufficient to restore clonal number and size to control levels (D, E). Confocal micrographs are projections of multiple sections, showing EADs 7 days AEL. Nuclei were counterstained with DAPI. Scale bars: 100 μm (D, F, H, J, L). ( N ) Quantification of clonal to total EAD volume ratios from confocal micrographs of mosaic EADs of the indicated genotypes. Data represent means ± SD, n = 7–16. Ordinary one-way ANOVA with Tukey's multiple comparisons test was used to determine significance, **** P < 0.0001, n.s. = non-significant. See also .

Article Snippet: Drosophila melanogaster coding DNA sequences of PIH1D1 (CG5792), Pontin (CG4003), SmD3 (CG8427), SmB (CG5352), SmG (CG9742), SmF (CG16792) and Ecd (CG5714) were amplified from respective cDNAs with Phusion HS II polymerase (ThermoFisher Scientific Cat# F549L) and cloned into the pENTR4 dual selection vector (ThermoFisher Scientific Cat# A10465).

Techniques: Mutagenesis, Functional Assay, In Vivo, Generated, Western Blot, Transfection, Control, Two Tailed Test, Clone Assay, Over Expression

Journal: American Journal of Translational Research

Article Title: Upregulation of SPI1 during myocardial infarction aggravates cardiac tissue injury and disease progression through activation of the TLR4/NFκB axis

doi:

Figure Lengend Snippet: SPI1 siRNA reduced hypoxia-induced damage in cardiomyocytes. (A, B) mRNA (A) and protein (B) levels of SPI1 in HL-1 cells after different time of hypoxic treatment determined by RT-qPCR and western blot analysis; (C) Apoptosis rate in HL-1 cells after hypoxic treatment examined by TUNEL assay; (D) mRNA and protein levels of SPI1 in HL-1 cells after si-SPI1 transfection quantified by RT-qPCR and western blot analysis; (E) Apoptosis rate in HL-1 cells after SPI1 silencing determined by TUNEL assay; (F) Production of IL-6 and TNF-α in the supernatant of SPI1 cells examined using ELISA kits. All data are presented as mean ± SD. For cellular experiments, repetition = 3. Differences were analyzed by the unpaired t test (C-E), one-way ANOVA (A and B) or two-way ANOVA (F). *P < 0.05 vs. the control group; #P < 0.05 vs. the si-NC group.

Article Snippet: Antibodies for western blot analysis Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) A one-step TUNEL kit (Beyotime) was used to examine apoptosis of cardiomyocytes.

Techniques: Quantitative RT-PCR, Western Blot, TUNEL Assay, Transfection, Enzyme-linked Immunosorbent Assay, Control

Journal: American Journal of Translational Research

Article Title: Upregulation of SPI1 during myocardial infarction aggravates cardiac tissue injury and disease progression through activation of the TLR4/NFκB axis

doi:

Figure Lengend Snippet: Overexpression of TLR4 activated the NFκB signaling pathway and blocked the protective roles of SPI1 siRNA. (A) SPI1 and TLR4 protein levels and the NFκB phosphorylation in HL-1 cells after si-SPI1 and oe-TLR4 transfection and hypoxia exposure determined using western blot analysis; (B) Apoptosis of HL-1 cells examined by the TUNEL assay; (C) Secretion of IL-6 and TNF-α in cells examined using ELISA kits; (D) Protein level of TLR4 and phosphorylation of NFκB in HL-1 cells after oe-TLR4 transfection and hypoxia exposure determined using western blot analysis; (E) Cell apoptosis examined by the TUNEL assay; (F) Secretion of the pro-inflammatory cytokines in cells examined using ELISA kits. For cellular experiments, repetition = 3. All data are presented as mean ± SD. Differences were analyzed by the unpaired t test (B and E) or two-way ANOVA (A, C, D, and F). *P < 0.05 vs. oe-NC; #P < 0.05 vs. the si-SPI1 + oe-NC group; @P < 0.05 vs. oe-NC.

Article Snippet: Antibodies for western blot analysis Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) A one-step TUNEL kit (Beyotime) was used to examine apoptosis of cardiomyocytes.

Techniques: Over Expression, Phospho-proteomics, Transfection, Western Blot, TUNEL Assay, Enzyme-linked Immunosorbent Assay

Journal: European journal of human genetics : EJHG

Article Title: Analysis of cilia dysfunction phenotypes in zebrafish embryos depleted of Origin recognition complex factors

doi: 10.1038/s41431-019-0338-0

Figure Lengend Snippet: A, Live images of 48 hpf zebrafish embryos. NI, uninjected controls, CTRL MO, embryos injected with a control MO, Orc1 MO, translation blocking MO against Orc1. Arrow indicates pericardial oedema. Scale bar: 500 µm.

Article Snippet: To generate a DIG-labelled antisense in situ probe for the detection of orc1 , the plasmid containing a fragment of the Orc1 coding sequence was linearized with HindIII and transcribed using T7 RNA polymerase (NEB, Frankfurt am Main, Germany) and DIG labelling mix (Roche, Mannheim, Germany).

Techniques: Injection, Blocking Assay

Journal: European journal of human genetics : EJHG

Article Title: Analysis of cilia dysfunction phenotypes in zebrafish embryos depleted of Origin recognition complex factors

doi: 10.1038/s41431-019-0338-0

Figure Lengend Snippet: A, Flat mounts of 4 and 8 ss embryos after orc1 ISH. Arrow indicates expression throughout the embryo, but also in the tailbud, where the KV forms. Scale bars: 150 µm.

Article Snippet: To generate a DIG-labelled antisense in situ probe for the detection of orc1 , the plasmid containing a fragment of the Orc1 coding sequence was linearized with HindIII and transcribed using T7 RNA polymerase (NEB, Frankfurt am Main, Germany) and DIG labelling mix (Roche, Mannheim, Germany).

Techniques: Expressing

Journal: European journal of human genetics : EJHG

Article Title: Analysis of cilia dysfunction phenotypes in zebrafish embryos depleted of Origin recognition complex factors

doi: 10.1038/s41431-019-0338-0

Figure Lengend Snippet: A, Heart looping in Orc1 morphants is rescued by co-injection with RNA encoding human ORC1. Embryos were scored after cmlc2 ISH. D, correctly looped heart, N, no loop, L, inversely looped heart.

Article Snippet: To generate a DIG-labelled antisense in situ probe for the detection of orc1 , the plasmid containing a fragment of the Orc1 coding sequence was linearized with HindIII and transcribed using T7 RNA polymerase (NEB, Frankfurt am Main, Germany) and DIG labelling mix (Roche, Mannheim, Germany).

Techniques: Injection

Journal: Marine Biotechnology (New York, N.y.)

Article Title: Induction of Autophagy by Amino Acid Starvation in Fish Cells

doi: 10.1007/s10126-012-9432-9

Figure Lengend Snippet: Amino-acid-starvation-induced autophagy in ZE cells expressing a fluorescent autophagosome marker. ZE cells were stably transfected with the plasmid encoding GFP–MAP1-LC3B fusion protein. a , b Immunofluorescence images of cells fixed after incubation for 6 h at 28.5°C in Leibovitz’s L-15 medium containing 2% fetal calf serum (FCS) ( a ) or in Hanks’ buffered salt solution (HBBS) ( b ). Bar = 10 μm. Representative cells containing GFP–MAP1-LC3B dots (i.e., autophagosomal structures) can be seen in panel b . c The transfected cells were treated with HBSS for 0 ( lane 1 ), 1 ( lane 2 ), 2 ( lane 3 ), 3 ( lane 4 ), 4 ( lane 5 ), and 6 h ( lane 6 ). The cell lysates were analyzed by Western blotting with anti-human MAP1-LC3B and anti-tubulin antibodies. Arrowheads indicate stained signals for MAP1-LC3B. Asterisk indicates the band of the degraded GFP–MAP1-LC3B products. Tubulin was detected as the internal control. d Time-dependent induction of autophagy in amino acid-starved cells was quantified by counting punctate structures under a fluorescence microscope as described in the “ ” section. e Supernatant ( lanes 1 and 3 ) and pellet ( lanes 2 and 4 ) fractions from ultracentrifugation (100,000 × g ) of normal transfectant homogenates ( lanes 1 and 2 ) and transfectant starved of amino acids for 6 h ( lanes 3 and 4 ) were analyzed by Western blotting using antibodies against GFP, aldolase (a cytosolic marker), and transferrin receptor (a membrane protein marker). GFP–MAP1-LC3B-I, GFP–MAP1-LC3B-II, aldolase, and the transferrin receptor are indicated by arrowheads . Each value shown represents the mean of three independent experiments. Error bars represent one standard deviation. * P < 0.01 vs. control

Article Snippet: Proteins were detected by Western blotting with primary antibodies against MAP1-LC3B (Medical & Biological Laboratories), GFP (Medical & Biological Laboratories), aldolase (Santa Cruz Biotechnology), and transferrin receptor (Santa Cruz Biotechnology); a horseradish peroxidase-conjugated secondary antibody (GE Healthcare); and an ECL Western Blotting Detection kit (GE Healthcare) according to the manufacturer’s instructions.

Techniques: Expressing, Marker, Stable Transfection, Transfection, Plasmid Preparation, Immunofluorescence, Incubation, Western Blot, Staining, Control, Fluorescence, Microscopy, Membrane, Standard Deviation

Journal: Marine Biotechnology (New York, N.y.)

Article Title: Induction of Autophagy by Amino Acid Starvation in Fish Cells

doi: 10.1007/s10126-012-9432-9

Figure Lengend Snippet: Effects of 3-methyladenine (3-MA) and wortmannin on amino-acid-starvation-induced autophagy in ZE cells stably transfected with GFP–MAP1-LC3B. a The class III phosphatidylinositol 3-kinase inhibitors (i.e., 3-MA and wortmannin) repressed amino-acid-starvation-induced autophagy. After ZE cells were treated with 3-MA (1 mM) or wortmannin (100 nM) for 1 h, they were amino-acid-starved by incubation in HBBS for 6 h at 28.5°C, fixed and visualized by fluorescence microscopy. b Effects of 3-MA and wortmannin on amino-acid-withdrawal-induced degradation of long-lived proteins in ZE cells. ZE cells were radiolabeled with 370-MBq/mL l -[U- 14 C]valine for 48 h at 28.5°C, incubated in the presence of 3-MA (1 mM), wortmannin (100 nM), or no inhibitor for 1 h, and then amino-acid-starved by incubation in HBBS for 6 h at 28.5°C. The cell medium and cell lysates were precipitated with trichloroacetic acid, and the ratio of acid-soluble radioactivity in the medium to the acid-precipitable radioactivity in the cell lysate was determined by liquid scintillation counting and used as the rate of long-lived protein degradation as described in the “ ” section. Each value represents the mean of three independent experiments. Error bars represent one standard deviation. Asterisks denote a statistical difference between nutrient rich and starvation conditions ( P < 0.01)

Article Snippet: Proteins were detected by Western blotting with primary antibodies against MAP1-LC3B (Medical & Biological Laboratories), GFP (Medical & Biological Laboratories), aldolase (Santa Cruz Biotechnology), and transferrin receptor (Santa Cruz Biotechnology); a horseradish peroxidase-conjugated secondary antibody (GE Healthcare); and an ECL Western Blotting Detection kit (GE Healthcare) according to the manufacturer’s instructions.

Techniques: Stable Transfection, Transfection, Incubation, Fluorescence, Microscopy, Radioactivity, Standard Deviation

Journal: Kidney international

Article Title: Podocyte cell cycle regulation and proliferation in collapsing glomerulopathies.

doi: 10.1046/j.1523-1755.2000.00149.x

Figure Lengend Snippet: Fig. 2. Podocyte phenotype in normal adult kidney and in early lesions of HIVAN. (Left panels; a–c) Normal adult kidney. Synapto- podin is strongly expressed in normal adult podocytes, at the level of foot processes (a). Nuclear expression of cyclin D1 (b) and p57 (c) is evident in podocyte. (Middle panels; d–f) CG, nonaffected glomerulus. Synapto- podin expression is markedly reduced in non- affected glomeruli of CG (d) with a weak punctuated staining compared with a strong linear staining seen in normal control (a). Cyclin D1 (e) expression is loss in dysregu- lated podocytes of nonaffected glomeruli where nuclear immunostaining for p57 is still present (f). (Right panels; g–i) CG (serial sec- tions), segmental collapse. While immuno- staining for synaptopodin (g) and cyclin D1 (h) is diffusely reduced or not detectable, loss of p57 expression (i) is restricted to the area of segmental collapse (arrowheads) but is pre- served in nonaffected capillaries of the same glomerulus. (a–c, g–i, 3100; d–f, 3120).

Article Snippet: Sections were METHODS then incubated overnight at 48C with monoclonal antibodSelection of cases ies to synaptopodin (1:25) [14, 15], p27 (1:100; Transduction Laboratories, Lexington, KY, USA), Ki-67 (1:1000;Ten cases of CG, including eight HIVAN and two idio- pathic CG, were selected from the archives of the Renal Novocastra Laboratories, Inc., Burlingame, CA, USA), and cyclin D1 (1:100; Santa Cruz Biotechnology, SantaPathology laboratories at Montefiore Medical Center and the University of Connecticut (Farmington, CT, USA).

Techniques: Expressing, Staining, Control, Immunostaining

Journal: Kidney international

Article Title: Podocyte cell cycle regulation and proliferation in collapsing glomerulopathies.

doi: 10.1046/j.1523-1755.2000.00149.x

Figure Lengend Snippet: Fig. 3. Altered podocyte cell cycle and prolif- eration in CG. Cyclin kinase inhibitor (CKI) expression including p57 (a) and p27 (b) is diffusely lost in globally affected glomeruli. Note that Bowman’s space is completely filled with podocytes. In areas of podocyte crowd- ing, cyclin A (c) and Ki-67 (i) expression is observed in podocytes (arrowheads in c and d; 3120). (a and b) Serial sections. (a–d) HIVAN.

Article Snippet: Sections were METHODS then incubated overnight at 48C with monoclonal antibodSelection of cases ies to synaptopodin (1:25) [14, 15], p27 (1:100; Transduction Laboratories, Lexington, KY, USA), Ki-67 (1:1000;Ten cases of CG, including eight HIVAN and two idio- pathic CG, were selected from the archives of the Renal Novocastra Laboratories, Inc., Burlingame, CA, USA), and cyclin D1 (1:100; Santa Cruz Biotechnology, SantaPathology laboratories at Montefiore Medical Center and the University of Connecticut (Farmington, CT, USA).

Techniques: Expressing