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: bioRxiv

Article Title: Impaired migration and metastatic spread of human melanoma by a novel small molecule targeting the transmembrane domain of death receptor p75 NTR

doi: 10.1101/2023.11.13.566904

Figure Lengend Snippet: (A) Schematic shows the experimental design for the assessment of a human melanoma xenograft lung metastasis model following treatment with Np75-4A22. (B) Live-monitoring of lung metastatic tumor growth of A875-NT melanoma cells using the Xenogen IVIS-200 luciferase imaging system. Bioluminescent imaging of luciferase expressing A875-NT melanoma cells (control) in the lungs of SCID mice was measured 1, 2 and 3-weeks following treatment with Np75-4A22 (200 mg/kg/day) or vehicle alone. Representative images taken 3 weeks following implantation are shown (left panel). Graph shows the average quantification of luciferase signal (N=8-10 mice/group; right panel). Shown is one representative experiment from N=3 independent experiments. Data are expressed as the mean ± SEM. Means that are significantly different between treatments as analyzed by Student t-test are indicated. *, p<0.05: **, p<0.01. (C) Histological analysis of lung tumor burden in SCID mice bearing A875-NT or A875-shp75 tumors 3 weeks following of treatment with Np75-4A22 or vehicle alone. Left panel shows representative images of formalin fixed paraffin embedded lung sections stained by immunohistochemistry with a human-specific nucleolin antibody (brown) to detect the presence of tumor cells. Sections were counterstained with hematoxylin (blue). Scale bar, 60μm. Right panel shows quantification of the number of cancer cells per lung tissue section. Five non-sequential lung sections, 100 μm apart, were counted and averaged for each mouse (N=8-10 mice per group). Data is expressed as the mean ± SEM. Means that are significantly different are indicated. A875-NT vehicle vs Np75-4A22 ** p=0.0179; A875-NT vs A875-shp75 # p=0.0367; A857-shp75 vehicle vs Np75-4A22 not significant (n.s.), one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Human-specific mouse polyclonal nucleolin antibody was from Abcam (#ab136649).

Techniques: Luciferase, Imaging, Expressing, Formalin-fixed Paraffin-Embedded, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: Impaired migration and metastatic spread of human melanoma by a novel small molecule targeting the transmembrane domain of death receptor p75 NTR

doi: 10.1101/2023.11.13.566904

Figure Lengend Snippet: p75 NTR immunostaining (red) in lung metastasis induced by A875-NT (A) or shp75-A875 (B) cells counter-stained for human nucleolin (green) and DAPI (white). Scale bar, 100μM.

Article Snippet: Human-specific mouse polyclonal nucleolin antibody was from Abcam (#ab136649).

Techniques: Immunostaining, Staining