Figure 5. Transcellular transport of ASN internalized by immortalized (A549) and primary hAEC concludes with their exocytosis at the basolateral cell surface. ( A ) TEM visualization of ASN agglomerates at the basolateral surface of A549 cells attached to a culture dish and ( B ) their appearance in the TIRF microscopy field after exocytosis (red) from A549 (plasma membrane patches labeled with Alexa-488 WGA, green). TIRF was set to an illumination depth of < 100 nm. ( C ) Analogous ASN exocytosis images at the basolateral surface of primary hAEC cells in TEM and ( D ) in the TIRF field. ( E ) Analysis of three independent time lapse videos (as in D ) demonstrate appearance of ASN in the TIRF plane starting at 10 min post their application to the apical plane of super confluent hAECs and the increase in far-red fluorescence intensity in consequence of labeled ASN exocytosis over time (n = 3). ( F ) This contrasts with analogous setup with PFA fixed cells (negative control) which do not allow for transfer of ASN to the basolateral plane within the equivalent time intervals. Scale bars: ( A , C ): 500 nm, ( B , D , F ): 5