Bright field and fluorescence microscopic analysis of CX+ and CX− tumor sublines (A), Fabry fibroblasts (B), and Daudi Burkitt's lymphoma cells (C).

<p>Cells were stained either with a relevant isotype-matched control antibody (isotype) or with an Hsp70 (cmHsp70.1-FITC, green) or Gb3 (CD77 plus Cy3-conjugated secondary antibody, red) specific antibody. The co-localization of Hsp70 and Gb3 is visualized in yellow as a merge of red and green in the lowest panel. Scale bar marks 20 µm. In comparison to CX+ and Fabry cells, the density of Gb3 on the cell membrane of Daudi cells is markedly increased, as indicated by a high mean fluorescence intensity of the CD77 staining. A double stain of Hsp70-FITC and Gb3-Cy3 indicates co-localization of both markers on the cell surface (lower right graph). Similar findings were obtained in experiments using the Colo+/Colo− tumor sublines (data not shown). On the right hand panel of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001925#pone-0001925-g004" target="_blank">Figure 4C</a>, representative flow cytometric profiles of Daudi cells are shown; the upper graph represents the double staining pattern of the isotype-matched antibodies IgM-PE and IgG1-FITC; the graphs below show single staining of Daudi cells with Hsp70-FITC (second graph) and CD77-PE (third graph). The fourth graph represents the double-staining pattern of Daudi cells using Hsp70-FITC and CD77-PE antibodies.</p

Flow cytometric analysis of viable, 7-AAD- and Annexin-V-negative CX+ tumor cells either untreated (Ctrl) or after treatment with a sublethal dose of 1 phenyl-2-hexadecanoyl amino-3-morpholino-1-propanol (PPMP1, 25 µM) for 3 or 6 days.

<p>Data are displayed as the mean fluorescence intensity of Hsp70 positive cells±SE from at least 3 independent experiments.</p>a<p>means significantly different from control (P<0.05).</p

Globotriaosylceramide content is significantly higher in Hsp70 membrane-positive tumor sublines compared to their negative counterparts.

<p>Comparative analysis of the cell surface density of glycosphingolipids such as Gb3 (globotriaosylceramide, CD77 Ab), LacCer (lactosylceramide, CDw17 Ab), DoCer (dodecasaccharideceramide, CD65s Ab, data not shown), GalCer (galactosylceramide, MAB 342 Ab), Cer (ceramide, MID15B4 Ab), and GM1 (Cholera Toxin subunit B) in CX+/CX− and Colo+/Colo− tumor sublines. *Indicates values for Hsp70 membrane-positive (CX+, Colo+) and the corresponding Hsp70 membrane-negative (CX−, Colo−) tumor subline that are significantly different (P<0.03).</p

Hsp70 predominantly interacts with artificial raft liposomes containing Gb3.

<p>Quantification of the amount of recombinant Hsp70 protein associated with unilamellar raft-like liposomal pellet fraction consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC)/brain sphingomyelin (SM)/cholesterol (Chol), (PC/SM/Chol, 17/50/33); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC)/brain sphingomyelin (SM)/cholesterol (Chol)/brain cerebroside sulfatide (CerSul) (PC/SM/Chol/CerSul, 17/45/33/5); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC)/brain sphingomyelin (SM)/cholesterol (Chol)/galactocerebroside (GalCer) (PC/SM/Chol/GalCer, 17/45/33/5); and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC)/brain sphingomyelin (SM)/cholesterol (Chol)/globotriasoylceramide (Gb3) (PC/SM/Chol/Gb3, 17/45/33/5). Data are the total amount of Hsp70 (ng) in the pellet fraction containing liposomes and are means (±SE) of 3 to 5 independent experiments.</p

Binding of Hsp70-FITC, and BSA-FITC to viable Gb3 membrane-negative K562 and Gb3 membrane-positive Daudi cells.

<p>(A) Significant binding of Hsp70-FITC was observed to Daudi cells at a concentration of 12 µg/ml and 24 µg/ml. (B) BSA-FITC did neither bind to K562 nor to Daudi cells, at any of the tested concentrations. (C) Specific blocking of Hsp70-FITC binding by anti-Gb3 monoclonal antibody CD77. Daudi cells were kept either untreated or incubated with anti-Gb3 (CD77), or anti-ceramide (MID15B4) antibodies (5 µg/ml) for 30 min on ice prior to incubation with Hsp70-FITC (60 µg/ml, 30 min on ice) and analysis by flow cytometry.</p

Quantification of cytosolic and plasma membrane-bound Hsp70 in CX+/CX− and Colo+/Colo− carcinoma sublines.

<p>Western blot analysis of biotinylated whole cell lysates (cytoplasm, upper graph), and plasma membranes (lower graph) of CX+/CX− and Colo+/Colo− tumor sublines. A corresponding Western blot was stained with the Hsp70 specific mAb cmHsp70.1. The figure shows a representative blot from three independent experiments. The Hsp70 surface phenotypes of the tumor sublines, as determined by flow cytometric analysis, are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001925#pone-0001925-t001" target="_blank">Table 1</a>.</p