Figure 3
Figure 3. Nucleolin associates with Fas on the surface of primary B-cell lymphoma cells. Localization of nucleolin (NCL)-Fas complexes in B-cell lymphomas. BJAB cells (A) a primary NHL (B) and PBMCs (C) were incubated with an anti-NCL antibody (MS-3), anti-Fas antibody (Abcam), and respective secondary antibodies stepwise at 4°C. Subsequent incubation with wheat germ agglutinin alexa 555 was followed by mounting with prolonged gold anti-fade reagent containing 4,6 diamidino-2-phenylindole (DAPI) and examination by confocal microscopy. The images were captured by the Nikon A1R confocal laser microscope system (Nikon Instruments). All images were acquired at similar voltages for Channel 1 (488 nm) 605-620 V and Channel 3 (647 nm) 510-517 V. An aberration corrected objective (Paplon 1:40) and Nomarski prism for Brightfield DIC image was used for acquiring images. Original image size, 1024 × 1024 with clip size of 302 × 280. PowerPoint was used for further image processing and all panels were adjusted for brightness at correction 44. Top panels (left to right): NCL staining on the surface of BJAB cells (green). Fas staining on the surface of BJAB cells (red). Wheat germ agglutinin revealing surface sialic acid-modified proteins (white) was used as a control for cell membrane localization. Bottom panels (left to right): brightfield image revealing whole cell structure. DAPI nuclear stain (blue). Merged/overlaid images of NCL, Fas, and DAPI; note an almost a complete colocalization of Fas and NCL throughout the surface of BJAB cells (A) and the primary NHL (B) (yellow) and no colocalization on the surface of a healthy lymphocyte, largely because of a lack of surface Fas and low levels of NCL (C). For colocalization staining, we selected a healthy PBMC that had slight positive NCL surface staining as 2 of 15 B-cells scanned by flow cytometry for surface NCL revealed a small shift in staining intensity (MFI) (data not shown) (D) Intensity profile and Pearson’s coefficient analysis for colocalization, revealing positive colocalized staining of Fas and NCL in primary NHL and BJAB cells (merge image from A-B).

Nucleolin associates with Fas on the surface of primary B-cell lymphoma cells. Localization of nucleolin (NCL)-Fas complexes in B-cell lymphomas. BJAB cells (A) a primary NHL (B) and PBMCs (C) were incubated with an anti-NCL antibody (MS-3), anti-Fas antibody (Abcam), and respective secondary antibodies stepwise at 4°C. Subsequent incubation with wheat germ agglutinin alexa 555 was followed by mounting with prolonged gold anti-fade reagent containing 4,6 diamidino-2-phenylindole (DAPI) and examination by confocal microscopy. The images were captured by the Nikon A1R confocal laser microscope system (Nikon Instruments). All images were acquired at similar voltages for Channel 1 (488 nm) 605-620 V and Channel 3 (647 nm) 510-517 V. An aberration corrected objective (Paplon 1:40) and Nomarski prism for Brightfield DIC image was used for acquiring images. Original image size, 1024 × 1024 with clip size of 302 × 280. PowerPoint was used for further image processing and all panels were adjusted for brightness at correction 44. Top panels (left to right): NCL staining on the surface of BJAB cells (green). Fas staining on the surface of BJAB cells (red). Wheat germ agglutinin revealing surface sialic acid-modified proteins (white) was used as a control for cell membrane localization. Bottom panels (left to right): brightfield image revealing whole cell structure. DAPI nuclear stain (blue). Merged/overlaid images of NCL, Fas, and DAPI; note an almost a complete colocalization of Fas and NCL throughout the surface of BJAB cells (A) and the primary NHL (B) (yellow) and no colocalization on the surface of a healthy lymphocyte, largely because of a lack of surface Fas and low levels of NCL (C). For colocalization staining, we selected a healthy PBMC that had slight positive NCL surface staining as 2 of 15 B-cells scanned by flow cytometry for surface NCL revealed a small shift in staining intensity (MFI) (data not shown) (D) Intensity profile and Pearson’s coefficient analysis for colocalization, revealing positive colocalized staining of Fas and NCL in primary NHL and BJAB cells (merge image from A-B).

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