Journal: Biomolecules

Article Title: Myogenic Anti-Nucleolin Aptamer iSN04 Inhibits Proliferation and Promotes Differentiation of Vascular Smooth Muscle Cells

doi: 10.3390/biom14060709

Figure Lengend Snippet: Nucleolin localization and iSN04 incorporation in A10 cells. ( A ) Representative fluorescence images of nucleolin staining of A10 cells in GM (day 0) and DM with or without 10 μM iSN04 (day 4). Scale bar, 50 μm. ( B ) Representative fluorescence images of A10 cells treated with 5 μg/mL 6-FAM-iSN04 in GM. Scale bar, 50 μm. 6-FAM, 6-carboxyfluorescein; DAPI, 4′,6-diamidino-2-phenylindole; DM, differentiation medium; GM, growth medium.

Article Snippet: The cells were fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100, and immunostained with 1.0 μg/mL rabbit polyclonal anti-nucleolin antibody (ab22758; Abcam, Cambridge, UK) or 1.0 μg/mL mouse monoclonal anti-α-SMA antibody (1A4, ab7817; Abcam) overnight at 4 °C.

Techniques: Fluorescence, Staining

Journal: Biomolecules

Article Title: Myogenic Anti-Nucleolin Aptamer iSN04 Inhibits Proliferation and Promotes Differentiation of Vascular Smooth Muscle Cells

doi: 10.3390/biom14060709

Figure Lengend Snippet: The effect of iSN04 on proliferation and differentiation of hAoSMCs. ( A ) Representative fluorescence images of nucleolin staining of hAoSMCs in hGM (day 0) and hDM (day 4). Scale bar, 50 μm. ( B ) Representative fluorescence images of EdU staining of hAoSMCs pre-treated with 30 μM iSN04 in hGM for 48 h and then with 10 μM EdU in hGM for 12 h. Scale bar, 200 μm. The ratio of EdU + cells were quantified. ** p < 0.01 vs. control (Student’s t -test). n = 4. ( C ) qPCR results of a cell-cycle marker gene, Ki-67 ( MKI67 ), and contractile SMC marker genes, α-SMA ( ACTA2 ), SM22α ( TAGLN ), and caldesmon ( CALD1 ), in hAoSMCs treated with 30 μM iSN04 in hGM for 48 h. * p < 0.05; ** p < 0.01 vs. control (Student’s t -test). n = 3. DAPI, 4′,6-diamidino-2-phenylindole; DM, differentiation medium; EdU, 5-ethynyl-2′-deoxyuridine; GM, growth medium; hAoSMC, human aortic smooth muscle cell; hDM, differentiation medium for human aortic smooth muscle cell; hGM, growth medium for human aortic smooth muscle cell; qPCR, quantitative real-time RT-PCR; SMC, smooth muscle cell.

Article Snippet: The cells were fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100, and immunostained with 1.0 μg/mL rabbit polyclonal anti-nucleolin antibody (ab22758; Abcam, Cambridge, UK) or 1.0 μg/mL mouse monoclonal anti-α-SMA antibody (1A4, ab7817; Abcam) overnight at 4 °C.

Techniques: Fluorescence, Staining, Control, Marker, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Proteomic Analysis of Chikungunya Virus Infected Microgial Cells

doi: 10.1371/journal.pone.0034800

Figure Lengend Snippet: CHME-5 cells either mock infected or infected with CHIKV at MOI 0.1 were collected at day 2 p.i. (A, B, D) or on days 1 to 3 p.i. (C) and subsequently (A) stained with an anti-alphavirus antibody and the percentage of infected cells analyzed by flow cytometry or (B) stained with Annexin V-FITC and PI and the percentage of apoptotic cells analyzed by flow cytometery or (C, D) used for total protein extraction and (C) analyzed by western blotting with an anti-alphavirus monoclonal antibody and an anti-actin polyclonal antibody or (D) the differential proteome determined by 2D-PAGE. Representative gels from 6 biological replicates are shown. (A and B) Bar graphs represent the means ± SD of 6 replications.

Article Snippet: Antibodies used included a 1∶400 dilution of a rabbit anti-JAK 2 polyclonal antibody (sc-278; Santa Cruz Biotechnology Inc.), a 1∶6,000 dilution of a rabbit anti-Hsp90 polyclonal antibody (sc-7947; Santa Cruz Biotechnology Inc.), a 1∶4,000 dilution of a rabbit anti-Hsp70 polyclonal antibody (sc-1060; Santa Cruz Biotechnology Inc.), a 1∶5,000 dilution of a rabbit anti-nucleolin polyclonal antibody (sc-13057; Santa Cruz Biotechnology Inc.), a 1∶3,000 dilution of a rabbit anti-heterogeneous nuclear ribonucleoprotein polyclonal antibody (ab65049; Abcam, Cambridge, MA), a 1∶10,000 dilution of a rabbit anti-elongation factor 2 polyclonal antibody (ab33523; Abcam) all followed by a 1∶4,000 dilution of a goat horseradish peroxidase (HRP) conjugated anti-rabbit IgG polyclonal antibody (31460; Pierce, Rockford, IL) as well as a 1∶5,000 dilution of a goat anti-actin polyclonal antibody (sc-1616; Santa Cruz Biotechnology Inc.) followed by a 1∶8,000 dilution of a rabbit HRP conjugated anti-goat IgG polyclonal antibody (31402; Pierce), as well as a 1∶1000 dilution of a mouse anti-alphavirus monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) followed by a 1∶5,000 dilution of a rabbit HRP conjugated anti-mouse IgG polyclonal antibody (A9044; Sigma, Sigma-Aldrich, St Louis, MO).

Techniques: Infection, Staining, Flow Cytometry, Protein Extraction, Western Blot

Journal: PLoS ONE

Article Title: Proteomic Analysis of Chikungunya Virus Infected Microgial Cells

doi: 10.1371/journal.pone.0034800

Figure Lengend Snippet: (A) CHME-5 cells were either mock infected or infected with CHIKV at MOI 0.1 before extraction of proteins and analysis by Western blot analysis on 1 and 2 d.p.i. hnRNP: heterogeneous nuclear ribonucleoprotein; NCL: nucleolin; JAK2: tyrosine-protein kinase JAK2; Hsp70: heat shock protein 70; Hsp90: heat shock protein 90. (B). CHME-5 cells were either mock infected or infected with CHIKV at MOI 0.1 before extraction of total RNA and analysis by RT-PCR on 1, 2 and 3 d.p.i. BRE1B: E3 ubiquitin-protein ligase; CUL9: Cullin-9; CHD2: chromodomain-helicase-DNA binding protein 2; MTERF: mitochondrial precursor transcription termination factor; ROD1: regulator of differentiation 1 isoform; PIK3CD: phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta; GCDH: mitochondrial glutaryl-CoA dehydrogenase isoform precursor; HSDL2: hydroxysteroid dehydrogenase-like protein 2; PLCH2: 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase eta-2; ALOX12: 12-lipoxygenase; DRPLA: Dentatorubral pallidoluysian atrophy protein; DENND3: DENN domain-containing protein 3; HIS1H2B: Histone 2B.

Article Snippet: Antibodies used included a 1∶400 dilution of a rabbit anti-JAK 2 polyclonal antibody (sc-278; Santa Cruz Biotechnology Inc.), a 1∶6,000 dilution of a rabbit anti-Hsp90 polyclonal antibody (sc-7947; Santa Cruz Biotechnology Inc.), a 1∶4,000 dilution of a rabbit anti-Hsp70 polyclonal antibody (sc-1060; Santa Cruz Biotechnology Inc.), a 1∶5,000 dilution of a rabbit anti-nucleolin polyclonal antibody (sc-13057; Santa Cruz Biotechnology Inc.), a 1∶3,000 dilution of a rabbit anti-heterogeneous nuclear ribonucleoprotein polyclonal antibody (ab65049; Abcam, Cambridge, MA), a 1∶10,000 dilution of a rabbit anti-elongation factor 2 polyclonal antibody (ab33523; Abcam) all followed by a 1∶4,000 dilution of a goat horseradish peroxidase (HRP) conjugated anti-rabbit IgG polyclonal antibody (31460; Pierce, Rockford, IL) as well as a 1∶5,000 dilution of a goat anti-actin polyclonal antibody (sc-1616; Santa Cruz Biotechnology Inc.) followed by a 1∶8,000 dilution of a rabbit HRP conjugated anti-goat IgG polyclonal antibody (31402; Pierce), as well as a 1∶1000 dilution of a mouse anti-alphavirus monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) followed by a 1∶5,000 dilution of a rabbit HRP conjugated anti-mouse IgG polyclonal antibody (A9044; Sigma, Sigma-Aldrich, St Louis, MO).

Techniques: Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Binding Assay

Journal: PLoS ONE

Article Title: Proteomic Analysis of Chikungunya Virus Infected Microgial Cells

doi: 10.1371/journal.pone.0034800

Figure Lengend Snippet: Values of fold change in response to CHIKV infection, significance and half life of selected proteins.

Article Snippet: Antibodies used included a 1∶400 dilution of a rabbit anti-JAK 2 polyclonal antibody (sc-278; Santa Cruz Biotechnology Inc.), a 1∶6,000 dilution of a rabbit anti-Hsp90 polyclonal antibody (sc-7947; Santa Cruz Biotechnology Inc.), a 1∶4,000 dilution of a rabbit anti-Hsp70 polyclonal antibody (sc-1060; Santa Cruz Biotechnology Inc.), a 1∶5,000 dilution of a rabbit anti-nucleolin polyclonal antibody (sc-13057; Santa Cruz Biotechnology Inc.), a 1∶3,000 dilution of a rabbit anti-heterogeneous nuclear ribonucleoprotein polyclonal antibody (ab65049; Abcam, Cambridge, MA), a 1∶10,000 dilution of a rabbit anti-elongation factor 2 polyclonal antibody (ab33523; Abcam) all followed by a 1∶4,000 dilution of a goat horseradish peroxidase (HRP) conjugated anti-rabbit IgG polyclonal antibody (31460; Pierce, Rockford, IL) as well as a 1∶5,000 dilution of a goat anti-actin polyclonal antibody (sc-1616; Santa Cruz Biotechnology Inc.) followed by a 1∶8,000 dilution of a rabbit HRP conjugated anti-goat IgG polyclonal antibody (31402; Pierce), as well as a 1∶1000 dilution of a mouse anti-alphavirus monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) followed by a 1∶5,000 dilution of a rabbit HRP conjugated anti-mouse IgG polyclonal antibody (A9044; Sigma, Sigma-Aldrich, St Louis, MO).

Techniques: Infection

Journal: iScience

Article Title: Ercc2/Xpd deficiency results in failure of digestive organ growth in zebrafish with elevated nucleolar stress

doi: 10.1016/j.isci.2022.104957

Figure Lengend Snippet: Perturbed rRNA synthesis results in nucleolar stress (A) Transmission electron microscopy images of intestinal endothelial cell (IEC) nuclei in ercc2/xpd mutants and siblings at 5 dpf. Arrowheads point to enlarged nucleoli. Scale bars, 1 μm. (B–D) DAPI staining and immunostaining of the nucleolar markers fibrillarin and nucleolin in IECs in ercc2/xpd mutants and siblings at 5 dpf. Areas of dashed boxes are magnified. Arrowheads point to enlarged nucleoli, arrows indicate the translocation of nucleolar proteins to the nucleoplasm. Scale bars, 20 μm. See also Figure S7 . (E) Schematic diagram showing stepwise processing of the pre-rRNA transcript. a-c corresponds to the rRNA intermediates. 5′ETS and ITS1 probes are indicated with green and red lines, respectively. ETS, external transcribed spacer; ITS, internal transcribed spacer. (F) Northern blot using 5′ETS and ITS1 probes to detect precursor forms of rRNA in ercc2/xpd mutants and siblings at 5 dpf. Methylene blue staining was used as a loading control. (G and H) Representative E-Bioanalyzer analysis and measurement of the 28S/18S rRNA ratio in ercc2/xpd mutants (M) and siblings (S) at 5 and 7 dpf. Data are presented as mean ± SD, Student’s t test, NS, non-significant.

Article Snippet: Rabbit polyclonal anti-Nucleolin , Abcam , Cat#ab22758; RRID: AB_776878.

Techniques: Transmission Assay, Electron Microscopy, Staining, Immunostaining, Translocation Assay, Northern Blot

Journal: iScience

Article Title: Ercc2/Xpd deficiency results in failure of digestive organ growth in zebrafish with elevated nucleolar stress

doi: 10.1016/j.isci.2022.104957

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Nucleolin , Abcam , Cat#ab22758; RRID: AB_776878.

Techniques: Recombinant, Labeling, DNA Labeling, Blocking Assay, SYBR Green Assay, Protease Inhibitor, Plasmid Preparation, Flow Cytometry, In Situ, Sequencing, Staining, Bicinchoninic Acid Protein Assay, Western Blot, Northern Blot, Software