Figure 5 from Claudin-4 Modulates Autophagy via SLC1A5/LAT1 as a Mechanism to Regulate Micronuclei

Effect of claudin-4 manipulation on the intracellular distribution of transporters of amino acids. We studied the association of claudin-4 with the amino acid transporters, SLC1A5 and LAT1 (which regulate autophagy) in HGSOC cells using confocal microscopy, immunoblotting, and flow cytometry. A, Top, confocal images showing the intracellular distribution of SLC1A5 and claudin-4 in HGSOC cells; bottom, kymographs (from z-stacks and dotted yellow squares) highlighting colocalization. A similar phenotype is shown for LAT1 and claudin-4 in (B). The Pearson’s correlation coefficient for SLC1A5 and claudin-4, and LAT1 and claudin-4 is shown in (C). D, Percentage of HGSOC cells with autophagic flux during L-glutamine withdrawal measured by flow cytometry (two-tailed unpaired t test and Mann–Whitney test; three independent experiments; significance, P < 0.05). E and F, show representative confocal images (maximum projections) of the intracellular distribution of SLC1A5 before claudin-4 overexpression (OVCAR8 claudin-4) and downregulation (OVCA429 claudin-4 KD and OVCAR3 claudin-4 KD) in HGSOC cells with and without L-glutamine, respectively. Graphs show mean and SEM, scale bar, 20 µm.