bba1  (R&D Systems)

Journal: Science Advances

Article Title: Two-in-one nanoparticle platform induces a strong therapeutic effect of targeted therapies in P-selectin–expressing cancers

doi: 10.1126/sciadv.adr4762

Figure Lengend Snippet: ( A and B ) dSTORM imaging and analysis showing PD-L1 localizations per square micrometer in EMT6 cells with higher concentrations of talazoparib (Tal) after 48 hours. Blue: TIRF microscopy cell membrane; red: TIRF microscopy PD-L1 and dSTORM microscopy PD-L1 representative images. Data represent mean ± SD ( N = 3). Scale bars, 10 μm. ( C ) Representative images of EMT6 3D spheroids cocultured with activated splenocytes, at a 1:100 ratio, respectively, following treatments with Tal and PD-L1i, separately or combined. Scale bars, 300 μm. ( D ) Quantification of mCherry % area. Spheroids were either untreated (UT), supplemented with splenocytes (UT + spleen), or treated with 1 μM Tal and 10 μM PD-L1i, separately or combined. Data represent mean ± SD ( N = 3, n = 10), and statistical significance was calculated using two-sided repeated-measures ANOVA ( P < 0.05). ( E and F ) Representative images and quantification of immunofluorescence staining of melanoma (E) Murine D4M.3A. Scale bars, 100 μm. (F) Patient-derived specimens. Scale bars, 10 μm. The nucleus was stained with DAPI (blue), and P-selectin was stained with Cy5-labeled antibody (SELP, cyan). Data represent mean ± SD; at least three fields were imaged from each specimen, N = 3. ( G ) P-selectin expression in BRCA-mutated EMT6 primary (left) and brain metastases (right). Nuclei are stained with DAPI in blue and P-selectin (SELP) in red. Scale bars, 100 μm.

Article Snippet: The slides were subsequently incubated with mouse anti-human E/P selectin (R&D, 1:30 dilution) for 1 hour, and then incubated with mouse-IgG ƙ BP-CFL 488 (catalog no. sc-516176, Santa Cruz Biotechnology, 1:20 dilution) for an additional 1 hour, followed by Hoechst fluorescent dye (1:5000) for an additional 10 min for nuclei counterstaining.

Techniques: Imaging, Microscopy, Membrane, Immunofluorescence, Staining, Derivative Assay, Labeling, Expressing

Journal: Science Advances

Article Title: Two-in-one nanoparticle platform induces a strong therapeutic effect of targeted therapies in P-selectin–expressing cancers

doi: 10.1126/sciadv.adr4762

Figure Lengend Snippet: ( A ) Representative images of time course internalization of Cy5-labeled NPs into D4M.3A 3D spheroids. Scale bars, 400 μm. ( B ) Time course quantification of Cy5 intensity within D4M.3A 3D spheroids. ( C ) Representative images of Cy5-labeled NPs internalization into D4M.3A 3D spheroids after 22 hours. The spheroids were preincubated with SELPi for 1 hour before NPs addition. Scale bars, 400 μm. ( D ) Representative Z-stack images of Cy5-labeled PLGA-PEG-GLY-(OSO 3 Na) 2 NPs internalization into the core of D4M.3A 3D spheroids after 24 hours, preincubated in the presence or absence of 10 μM SELPi. Scale bars, 200 μm. ( E ) Quantification of Cy5 intensity over time of nontargeted NPs and P-selectin–targeted NPs, after preincubation of the D4M.3A 3D spheroids with increasing SELPi concentrations. All quantifications are representative of three independent experiments. The data correspond to the mean ± SD of at least eight 3D spheroids per group, and statistical significance was determined using a two-way ANOVA test. ( F and G ) P-selectin binding assay of PLGA-PEG-GLY-(OSO 3 Na) 2 NPs and PLGA-PEG NPs. Plates were coated with human recombinant P-selectin (rhSELP) or with skim milk (nonspecific binding control) and then incubated with PLGA-PEG-GLY-(OSO 3 Na) 2 NPs or PLGA-PEG NPs for 15 min. (F) Fluorescence intensity of Cy5-labeled NPs after incubation with rhSELP or skim milk. (G) The fluorescence intensity ratio of rhSELP/skim milk. Bars represent mean ± SD, and a two-way ANOVA was used for statistical analysis.

Article Snippet: The slides were subsequently incubated with mouse anti-human E/P selectin (R&D, 1:30 dilution) for 1 hour, and then incubated with mouse-IgG ƙ BP-CFL 488 (catalog no. sc-516176, Santa Cruz Biotechnology, 1:20 dilution) for an additional 1 hour, followed by Hoechst fluorescent dye (1:5000) for an additional 10 min for nuclei counterstaining.

Techniques: Labeling, Binding Assay, Recombinant, Control, Incubation, Fluorescence