Figure 3. The ASN uptake by AEC combines mechanistic features of caveolar endocytosis with macropinocytosis. Structured Illumination Microscopy shows the proximity of elements for caveolin mediated endocytosis and macropinocytosis during ASN uptake. ( A , B , E ) Upon the hAEC-internalized ASN (red) are associating with both caveolin-1 (white, A and B ) and macropinocytosis-markers Rank-5 (green, only B ) and C-terminal Binding Protein 1 (CtBP1; blue E) ( F ) enlarged view of E. Knocking down by siRNA interference Cav-1 ( C ), Rank-5 ( D ) or CtBP1 ( G ), each reduced ASN uptake in A549 and hAEC by over 50% (mean from 100 cells ± SD, n = 5, **p < 0.01). ( H ) Macropinocytosis enhancer phorbol-12-myristate-13-acetate (PMA) nearly doubled ASN uptake while macropinocytosis-inhibitor amiloride reduced uptake by half in A549 cells. ASN uptake is expressed as MFI increase within a confocal slice of the cell that internalized ASN compared with untreated control. Charts show individual data points, mean from 100 cells ± SD, n = 5, **p < 0.01. Scale bars: ( A , B , E ): 1 µm, ( F ): 500