Figure 8. Neutral sphingomyelinase activity does not participate in Hc-TeTx internalization in PC12 cells.

A) Undifferentiated (PC12) or NGF-differentiated PC12 (PC12NGF) cells were incubated Hc-Alexa 488 (1 nM) inside a thermostatic chamber at 37°C with CO₂ supply and coupled to a confocal microscopy. The internalization was followed by time-lapse image capturing for 90 min. Hc-TeTx binds with much more avidity to PC12NGF than to PC12, as can be seen in the 30 min image, but internalization at this time point is not detectable in any case. At 90 min a similar degree of Hc internalization can be observed in both undifferentiated and differentiated cells. B) The same experiment was conducted with prior incubation of the cells with 20 µM GW4869 for 1 h. The results observed are not significantly different from the obtained in the absence of GW4869, indicating that nSMase activity and Hc internalization are independent events. Magnifications of the highlighted area are shown on the right, in all the cases, corresponding to the 90 min time point.