Epifluorescent micrographs illustrating ZO-1 integrity in Caco-2 cell monolayers.

<p>Monolayers grown in DMEM (A), and exposed to <i>B. pilosicoli</i> 95/1000 for 6 h (B). In the control cells the ZO1 distribution is regular and limited to the junctions, which are intact. After 6 h incubation with <i>B. pilosicoli</i> the tight junctions are disrupted and the ZO-1 is punctuated and has migrated towards the cytoplasm (arrow). Photographs taken at a magnification of X 100.</p

Epifluorescent micrographs showing Hoechst staining of DNA in Caco-2 cells.

<p>Monolayers either grown in DMEM (A), or exposed to a culture of <i>B. pilosicoli</i> 95/1000 (B) for 6 h. Exposure to <i>B. pilosicoli</i> has resulted in many nuclei appearing condensed, and some showing clear chromatin fragmentation, consistent with apoptosis (arrows). Photographs taken at a magnification of X 100.</p

Scanning electron micrographs of <i>B. pilosicoli</i> interacting with Caco-2 cells.

<p>The cells were incubated for 6 h with DMEM (A), <i>B. pilosicoli</i> 95/1000 for 2 h (B), and 6 h (C), and WesB for 6 h (D). The non-infected cells show intact tight junctions with clear boundaries. After 2 h, <i>B. pilosicoli</i> 95/1000 mainly colonizes the cell boundaries (arrows), but by 6 h most of the cell surface is covered with spirochetes. The ends of the WesB cells can be seen penetrating the membrane of the Caco-2 cells (arrows), with the rest of the spirochete cell body lying on the Caco2 cell surface. The photographs were taken at magnifications of X 2,100 for panels A, B and C, and X 9,800 for panel D.</p

Transmission electron micrographs of <i>B. pilosicoli</i> interacting with Caco-2 cells.

<p>Cross-sections and tangential-sections of <i>B. pilosicoli</i> can be seen at the cell junctions (A) and under the cell membranes (B) (arrows). Spirochete cells can be seen attached to the Caco-2 cell surface (C), and invaginating into pit-like structures (arrow) in the Caco-2 cell membrane (D). Compared to the nuclei of control cells (E), the nuclei of many cells in the infected monolayers show chromatin condensation and fragmentation (arrows), consistent with apoptosis (F). The photographs were taken at magnifications of X 5,800, 7,900, 33,800, 24,500, 5,800 and 5,800, respectively.</p

Density of attachment of <i>B. pilosicoli</i> cells to Caco-2 cells after 2, 4 and 6 h incubation.^*

*<p>Results are derived from 12 fields of view. They are median (and the range) at each time point, where 0 represents no attachment observed and 5 represents the entire surface of the field covered with spirochetes such that the Caco-2 cell surface was not visible.</p

Epifluorescent micrographs showing actin staining in Caco-2 monolayers.

<p>Monolayers either grown in DMEM (A), or exposed to <i>B. pilosicoli</i> 95/1000 (B) for 6 h. In the control section there is regular distribution of FITC (phalloidin) over the monolayers. After 6 h incubation with <i>B. pilosicoli</i> the actin filaments are clearly mobilized and can be seen as round bodies on the junction of the Caco2 cells (arrows). Photographs taken at a magnification of X 100.</p

Effect of ephrin-A1-Fc on expression and phosphorylation of eNOS and Akt in HUVECs. HUVECs were exposed to ephrin-A1-Fc (1 µg/ml) for 0 h, 8 h, 24 h.

<p>Immunofluorescence (A), Real-time PCR (B) and Western blot analysis (C) demonstrated that ephrin-A1-Fc had no effect on eNOS expression of HUVECs (#, <i>P</i>>0.05, n = 3). D: Western blots demonstrating that P-eNOS_Ser1177 and P-Akt_Ser473 were up-regulated under ephrin-A1-Fc stimulation in a time-dependent manner. E: Quantity analysis of P-eNOS_Ser1177. F: Quantity analysis of P-Akt_Ser473. (*, <i>P</i><0.05, n = 4).</p

Schematic representation of signaling cascade involved in hypoxia-inducible ephrin-A1 modulation of angiogenesis in tumor hypoxic microenvironment.

<p>Schematic representation of signaling cascade involved in hypoxia-inducible ephrin-A1 modulation of angiogenesis in tumor hypoxic microenvironment.</p

Hypoxia up-regulated ephrin-A1 expression and soluble ephrin-A1 secretion in cancer cells.

<p>A: Western blots demonstrating that hypoxia elevated membrane bound ephrin-A1 expression in SCC-9 cells. B: Western blots demonstrating that hypoxia up-regulated soluble ephrin-A1 in supernatants of SCC-9 cells. SCC-9 cell density at 70–80% confluence was taken as 0 h when fresh culture medium was added. C: Cell rounding assay demonstrating that soluble ephrin-A1 in CM could activate EphA2 in U-251 GBM cells. Ephrin-A1(S), soluble ephrin-A1; N, normoxia conditioned medium group; H, hypoxia conditioned medium group.</p

PI3K/Akt mediated ephrin-A1-induced P-eNOS_Ser1177 in HUVECs.

<p>A: Representative Western blots for P-eNOS_Ser1177 and P-Akt_Ser473 from HUVECs that were starved in 0.1%BSA EBM-2 overnight and stimulated with ephrin-A1-Fc (1 µg/ml) for 30 min alone or together pre-treated with LY294002. B: Quantity analysis of P-Akt_Ser473. C: Quantity analysis of P-eNOS_Ser1177. (*, <i>P</i><0.05, n = 4) (#, <i>P</i>>0.05, n = 4).</p