Journal: Frontiers in Immunology

Article Title: Hyaluronic Acid Present in the Tumor Microenvironment Can Negate the Pro-apototic Effect of a Recombinant Fragment of Human Surfactant Protein D on Breast Cancer Cells

doi: 10.3389/fimmu.2020.01171

Figure Lengend Snippet: Apoptosis induction in BT20, BT474, and SKBR3 cell lines following HA challenge in the presence and absence of rfhSP-D (A) . The data were expressed as the mean of three independent experiments ( n = 3). A significant difference was seen among treated and untreated samples, as made evident by the shift in the fluorescence intensity. Staurosporine (1 μM/ml) was used as a positive control. Proliferative effects of rfhSP-D treatment on BT474 and SKBR3 breast cancer cell lines (B) . BT474 and SKBR3 cells were seeded in wells pre-coated with HA, HA + rfhSP-D, and rfhSP-D alone. The percentage of proliferative cells was evaluated by staining with mouse anti-human KI-67 antibody, and KI-67-stained cells were measured via flow cytometry. The data were generated from at least three independent experiments ( n = 3) and presented as mean ± SD (* p < 0.1, ** p < 0.01, and **** p < 0.0001). The statistical analysis was performed between rfhSP-D and HA + rfhSP-D-treated breast cancer cells. The secretion levels of FL-SP-D were confirmed and analyzed via western blotting (C) . Culture medium collected from BT20, BT474, and SKBR3 cell lines was passed through a maltose Agarose column, and the eluted fractions were validated via western blotting; FL-SP-D was detected at ~43 kDa only for BT474. Both BT20 and SKBR3 cell lines did not secrete any FL-SP-D. Secreted FL-SP-D by BT474 was tested for its ability to induce apoptosis (D) . No effect of secreted FL-SP-D was seen in terms of cell viability and apoptosis induction.

Article Snippet: The wells were washed with PBS, and 0.4 × 10 6 cells were added to the HA/rfhSP-D-coated wells and incubated 37°C for 24 h. For proliferative studies, the cells were washed with PBS and incubated with anti-mouse Ki-67 (BioLegend) diluted in a permeabilization reagent of the FIX&PERM kit (Fisher Scientific) for 30 min at RT.

Techniques: Fluorescence, Positive Control, Staining, Flow Cytometry, Generated, Western Blot

Journal:

Article Title: Immortalization and characterization of lineage-restricted neuronal progenitor cells derived from the porcine olfactory bulb

doi: 10.1016/j.jneumeth.2008.01.028

Figure Lengend Snippet: Immunocytochemical characterization of OBGF400 cells. A-D, F and H: Fluorescent microscopic images of immature OBGF400 cells after incubation with primary antibodies recognizing one of the neuron- or progenitor-specific markers TuJ1 (A, 10X objective), SOX2 (B, 40X objective), Nrg1 (C, 20X objective) or DXC (D, 20X objective) as well as the actin cytoskeleton filament (F, 40X objective) or the cell proliferation marker, Ki-67 (H, 40X objective) and subsequently exposed to FITC-labeled secondary antibody. E and G: Fluorescent microscopic images (40X objective) of phenotypically mature OBGF400 cells after incubation with primary antibodies recognizing either TuJ1 (E) or the actin cytoskeleton filament (G). I: The isotypic control, normal mouse IgG1 (20X objective). Size marker in panel C represents 100 μm.

Article Snippet: These primary antibodies were diluted in PBS containing 0.1% Triton-X-100 as follows: mouse anti-β-III-tubulin (TuJ1; 1:1000; Chemicon International, Inc.), rabbit anti-neuregulin-1 (Nrg1; 1:100; Abcam, Cambridge, MA), goat anti-doublecortin (DCX; 1:50; Santa Cruz Biotechnologies Inc., Santa Cruz, CA), goat anti-SOX2 (1:50; Santa Cruz Biotechnologies Inc.), mouse anti-Ki-67 (1:50; BD Biosciences, San Jose, CA), mouse anti-glial fibrillary acidic protein (GFAP; 1:500; Chemicon International, Inc.), mouse anti-2′3′cyclic nucleotide-3′-phosphodiesterase (CNPase; 1:100; Abcam), mouse anti-A2B5 (1:100; Chemicon) and mouse anti-actin (undiluted, BioGenex, San Ramon, CA).

Techniques: Incubation, Marker, Labeling