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. 2018 May 31;7:e34469. doi: 10.7554/eLife.34469

Figure 2. Transport of short-chain GM1 ceramides occurs via transcytosis.

Figure 2.

(A) Transport of C6:0 and C12:0 GM1-peptide fusions across T84 cell monolayers reported as apparent permeability (PAPP) shows that minimal transport occurs during a 4°C temperature block. (B) Analysis of MDCK-II monolayers loaded with 0.5 μM C6:0-GM1 peptide fusion in the absence (left and middle panels) or presence (right panel) of 2 mM EDTA. Live cell confocal imaging shows minimal transport occurs during a 10°C temperature block (middle panel) consistent with transcellular transport by membrane trafficking and minimal paracellular leak. In the presence of disrupted tight junctions (i.e. in the presence of EDTA, right panel) basolateral membranes are stained by paracellular passive diffusion (transcellular leak). (C) Transcytosis across MDCK-II monolayers is blocked by dynamin inhibition of endocytosis (50 μM Dyngo-4A). In Dyngo-4A treated cells (gray bars), there is a significant decrease in transepithelial transport of both C6:0 (n = 8) and C12:0 (n = 8) GM1-peptide fusions but not the unfused reporter peptide (n = 6). (D) Transcellular transport of the C6:0-GM1 peptide fusion (n = 6) or unfused reporter peptide (n = 5) in cell monolayers depleted of the exocyst complex by esiRNA transfection against EXO2 (gray bars). (mean ± s.e.m) (ns = non significant, **p<0.01, ****p<0.0001; Bonferroni’s multiple comparison test).