Fig. 3

CLDN6 increases WIP expression to regulate actin cytoskeleton-mediated autophagy. A Western blot showed levels of WIP protein in MCF-7 and MDA-MB-231 cells with or without CLDN6 overexpression. B Western blot showed levels of WIP protein in MCF-7/CLDN6 and MDA-MB-231/CLDN6 cells with or without WIP knockdown. C F-actin labeled with Rhodamine phalloidin in MCF-7/CLDN6 and MDA-MB-231/CLDN6 cells with or without WIP knockdown, zoomed-in image (right). Scale bar: 20 μm. D Western blot showed levels of autophagy-related proteins in MCF-7/CLDN6 and MDA-MB-231/CLDN6 cells with or without WIP knockdown. Results were from three independent experiments. E IF analysis showed LC3 positive cells in MCF-7/CLDN6 and MDA-MB-231/CLDN6 cells with or without WIP knockdown, zoomed-in image (right). Scale bar: 50 μm. F TEM analysis was performed on MCF-7/CLDN6 and MDA-MB-231/CLDN6 cells with or without WIP knockdown. Control group displayed several autophagic vacuoles (red arrowheads) which were not observed in WIP knockdown cells. Scale bar: 2 μm (top) and 1 μm (bottom). G The potential LIR motifs of the WIP. H The interaction of WIP and LC3 was detected by Co–IP assay in MDA-MB-231/CLDN6 cells. I IF analysis was performed to detect the colocalization of WIP (green) with LC3 (red) in MCF-7/CLDN6 and MDA-MB-231/CLDN6 cells. Scale bar: 20 μm. J Western blot showed the expression of LC3 and p62 in MCF-7/CLDN6, MDA-MB-231/CLDN6, MCF-7/CLDN6-shWIP and MDA-MB-231/CLDN6-shWIP cells treated with or without 100 nM Jasplakinolide for 2 h. Results were from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 and ns: no significance