Fig. 4
A–D Confocal images showing the actin filaments (F-actin) of the cytoskeleton of MCF7 cells grown on glass coverslips (Control, A) or PAN/G10 membranes for 24 (B), 48 h (C) or 72 h (D). Note the cellular aggregation induced by PAN/G10 forming cellular islets that grow significantly in size over time (72 h). E, F RT-qPCR determination of ID1 (E), KRT18, TJP1, EPCAM, CTNNB1, ACTB (F) gene expression in MCF7 cells RNA extracts grown on glass coverslips (Ctrl) or on PAN/G10 membranes as indicated. Bars represent means ± SD of three independent experiments. G, H Immunofluorescence and confocal microscopy images showing E-cadherin expression in cell membrane of MCF7 breast cancer cells grown on control coverslips or on PAN/G10 membranes. Note how the increased signal of E-cadherin at the edges of MCF7 cells forming islets due the PAN/G10 induced formation of adherens junctions. I The confocal image shows a detail of the cellular edge from two cells co-stained for E-cadherin (red signal) and Phalloidin-FITC (green signal), to reveal F-actin. The plots represent the linear profile of fluorescence intensity signals of E-cadherin (red) and F-actin (green) at the indicated points (a, b) in the image. Note the co-localization and similar intensity of both signals indicating the presence of intercellular E-cadherin-positive adherens junctions that normally are enriched in actin filaments (F-actin). J Western blotting analysis showing upregulation of E-cadherin expression in MCF7 cells grown on normal Petri dishes (Ctrl) or on PAN/G10 membranes. Histone H3 was used as loading control. The relative fold-increase of E-cadherin was calculated using Image J software. K, L Transmission electron micrographs of MCF7 cells grown on PAN/G10 membranes. Note, in panel (K), the normal appearance of the typical cellular organelles such as mitochondria (Mi), endoplasmic reticulum (ER), Golgi apparatus (Go), as well as the lack of heterochromatin clumps in the nucleus (Nu), of the epithelial-like cells which form cellular islets. The higher magnification detail in (L), shows a typical intercellular adherens junction (arrows). ***p < 0.0005, **p < 0.005, *p < 0.05, n.s not significant. Scale bar: 15 μm (A–D and G, H), 3 µm (K) and 500 nm (L)