RBD uptake is sensitive to CG pathway inhibitors.

A, B: AGS cells were pulsed with RBD, dextran and transferrin for 10 minutes and imaged at high resolution after fixation. Images in A and quantification in B shows that dextran and RBD are more correlated compared to dextran and transferrin (p-value < e-04) or transferrin and RBD (p-value < e-05) as measured using Pearson’s correlation coefficient (PCC). Number of cells = 10. C, D: AGS cells were treated with Control (0.6% DMSO) or AN96 25μM for 30 minutes, pulsed with RBD, dextran and transferrin for 30 minutes with Control or AN96 and imaged at high resolution upon fixation. Images are shown in C and quantification of Manders’ co-occurrence coefficient is shown in D. This depicts the fraction of RBD endosomal intensity with transferrin or dextran (i), the fraction of transferrin endosomal intensity with dextran or RBD (ii) and the fraction of dextran endosomal intensity with transferrin or RBD (iii). As seen in D(i), in control cells, the fraction of RBD endosomal intensity is more associated with dextran than transferrin (p-value < e-07). With AN96, internalized RBD and dextran is associated more with transferrin compared to control cells. Numbers of cells in each condition >10. p-value table is indicated in S1 Table. E, F: AGS cells were treated with Control or ML141 50μM for 30 minutes and pulsed with RBD and Dextran for 30 minutes with or without the inhibitor. RBD (p-value < e-9) and Dextran (p-value < e-20) uptake is significantly reduced upon treatment with ML141. Images are shown in E and quantification in F. Numbers of cells > 100 for each treatment. G, H: AGS cells were treated with Control (0.2% DMSO) or Amiloride 1mM for 30 minutes and pulsed with RBD, transferrin and dextran for 30 minutes with or without the inhibitor. RBD (p-value = 0.05), Dextran (p-value = 0.04) and transferrin (p-value = 0.013) uptake is not altered with Amiloride. Images are shown in G and quantification in H. Numbers of cells > 80 for each treatment. I: AGS cells were serum starved and treated with Control (0.2%DMSO), PMA alone (100nM), Amiloride alone (1mM) or in combination and pulsed with dextran for 30 minutes. Dextran uptake is enhanced with PMA; co-treatment with Amiloride abolishes this increase. Data representation is as described in Fig 1. Scale bar: 10 μm (A, C) and 40μm (E, G).

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