shifting), the CTB and SVLP were seen together, distributed about the cell membrane (Fig. 3A). Despite a clear association between the CTB and SVLP signals, there were also sites of SVLP interaction without any detectable CTB. The lower panels of Fig. 3A are zoomed on to the white square shown in the upper panel ‘‘Merge’’. Therein, association of the SVLP and CTB signals can be seen, as well as areas of red SVLP devoid of a green CTB signal. Moreover, the lower panel shows a different z-section to that in the upper panel, to demonstrate that sections showing apparently no associating (as in the white square of the upper panel) can display association in a different z-section (lower panels). Such observations that not all SVLP binding can be observed associated with sites of CTB binding may reflect additional SVLP interaction with the cell membrane at sites devoid of GM1, or a disparate distribution of the CTB to the GM1 sites. Within 10 min at 39uC, clear polarisation of the CTB together with the SVLP was observed (Fig. 3B), reminiscent of accumula- tion at the leading edge of the cell. There were also indications that CTB plus SVLP accumulation could occur at sites of intercellular contact (Fig. 3B, arrow head in upper panel). An apparently parallel internalisation of CTB with SVLP was visible at polarisation sites (Fig. 3B, arrow in lower panel). This accrual of SVLP at polarised sites related to the observations showing SVLP accumulating at sites of lamellipodia and filopodia formation (Fig. 3C, arrows). The formation of this leading edge was independent of SVLP binding, being observed in control DC cultures upon shifting from ice to 39uC (data not shown). At the time of observed maximum internalisation (maximum PK-resistance; see Fig. 2D), the SVLP remained associated with CTB (Fig. 3D). Although there were SVLP-containing structures in which CTB was absent, co-localisation algorithm analysis confirmed a high degree of co-localisation between the two (Fig. 3D, E). Such frequent association of SVLP with CTB indicated lipid raft involvement in DC uptake of SVLP. An important lipid raft-dependent endocytic route involves caveolar uptake. However, this appeared not to be involved, because no co- localisation with caveolin-1 was detectable (data not shown). Influence of cholesterol depletion on SVLP binding to DC SVLP association with lipid rafts was confirmed using antibody against CD9, due to the report of CD9 in lipid rafts of human monocytes [22]. DC were incubated as before with SVLP and CTB for 30 min on ice, but now also with antibody against CD9. The cells were then shifted to 39uC for 2 hrs. SVLP were observed associated with both CD9 and CTB (Fig. 4A, arrows). These results confirmed that SVLP primarily associated with lipid rafts; although not all SVLP were visualised together with CTB, they did associate with at least CTB or CD9. Considering that lipid rafts are cholesterol-rich membrane regions, the importance of cholesterol in SVLP uptake employed MBCD to deplete cholesterol. DC were treated with MBCD for 20 min at 39uC, followed by washing to remove MBCD and chilling on ice. SVLP binding (20 min on ice, in the absence of any further addition of MBCD) was clearly impaired (Fig. 4B, ‘‘No PK’’, ‘‘0’’ time point); statistical analysis showed the difference between untreated and MBDC-treated cells to be p,0.001. As expected, any SVLP still binding was on the cell surface, confirmed by its sensitivity to PK-treatment (Fig. 4B ‘‘PK- treated’’). Fig. 4B shows the effect of 20 mM MBCD; similar results were obtained with 10mM and 40mM MBCD (data not shown). The cultures were again washed to remove unbound SVLP, and fed with pre-warmed medium (39uC) containing 2 mM MBCD to maintain the cholesterol depletion, and shifted DC Endocytosis of SVLP back to 39uC. The gradual uptake of SVLP with time (time- dependent increase in PK-resistance at 39uC) was also significantly (p,0.001) impaired (Fig. 4B, ‘‘No PK’’ compared with ‘‘PK- treated). MBCD-treated and untreated cells, followed by addition of SVLP for 20 min on ice, were overlayed for analysis. Thereby, the reduction in the SVLP signals was clearly observable (Fig. S2A). Interestingly, the SVLP signal was not abrogated following MBCD treatment (also seen in Fig. 1B ‘‘No PK’’, ‘‘0’’). Accordingly, the analyses were repeated, but using CTB in place of SVLP. In this case, there was no apparent effect of the MBCD treatment (Fig. S2B). The MBCD pre-treatment followed by SVLP addition on ice was also repeated, but with a 10-fold higher concentration of SVLP (10 mg compared with 1 mg). With this 1 mg sample, the SVLP signal was somewhat weaker (Fig. S2C, left panel, compared with Fig. S2A, upper left panel). Nevertheless, the inhibitory effect of MBCD pre-treatment was still observable (Fig. S2C, second and third panels). The signal obtained with the 10 mg SVLP was much stronger (Fig. S2C, right panel, grey/black unshaded histograms), but now the MBCD did not reduce had little influence on the signal obtained. This higher concentration of SVLP gave a double peak of fluorescence intensity, whereas the MBCD showed inly a single peak, but of the triplicate samples there was no significant reduction in the SVLP signal. We did not attempt to increase the MBCD concentration, due to the increase toxicity for the cells. Indeed, the 20mM MBCD treatment led to increased cell membrane permeability (Fig. S2D middle panel), which could not be restored (Fig. S2D right panel). For these reasons, we can only interpret the effect of the MBCD on SVLP binding. Overall, it is difficult to interpret the influence of MBCD beyond the removal of cholesterol. Indeed, the major source of information on SVLP interaction with DC is provided by the colocalisation studies of SVLP with CTB and CD9 (Fig. 4A). Fate of SVLP in DC and involvement of intracellular organelles and compartments The internalisation of SVLP into cytoplasmic and membrane fractions raised the question of which endocytic processes and intracellular compartments were involved, and in particular if more than one pathway was operative. With primary DC and monocyte-derived DC, DNA plasmid transfection is highly inefficient [23], rendering analyses of intracellular organelle activity difficult by such means. Consequently, these analyses employed ligands and antibodies to identify various intercellular structures with which the SVLP associated. (i) Early endosomes. EEA-1 positive endosomal structures interacted closely with vesicles carrying SVLP, already clearly visible by 10 min (Fig. 4C). The 3-dimensional images showed EEA-1+ structures apparently ‘‘engulfing’’ the SVLP-containing vesicles (Fig. 4D). Interaction with early endosomes was confirmed using SVLP over-conjugated with Alexa488 to give a self-quench- ing of the fluorochrome and absence of signal. Once enzymatic degradation of these SVLP occurred, as in acidifying compart- ments containing the appropriate cathepsins, the fluorochrome signal should become detectable. A time-dependent appearance of the signal was indeed noted (Fig. 5A). Interestingly, no signal was detectable during first 30 min of incubation at 39uC (Fig. 5A), despite the appearance of unquenched SVLP associating the EEA- 1+ structures within 10 min (Fig. 4C). By 1 hr, a signal was now consistently apparent from quenched SVLP, seen in what appeared to be vesicular structures. The latter were primarily peripheral within the cell, and often associated with EEA-1+ structures, becoming more frequent at 2 hrs (Fig. 5A). PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e43248