X40 oil immersion objective

The samples on the nanofabricated coverslip were fixed with ice-cold 4% paraformaldehyde for 20 min, washed two times with phosphate-buffered saline (PBS), and permeablized with 0.1% Triton X-100 in PBS for 5 min. After washing with PBS, cultures were blocked with 10% goat serum for 1 h, and then incubated with primary antibodies against Vinculin (1:200, Sigma-Aldrich), E-cadherin (1:200, Cell Signaling, 3195), phospho-Myosin Light Chain (1:200, Cell Signaling, 3674), γ-tubulin (1:100, Abcam, ab11321), YAP (1:100, Cell Signaling, 4912 and Santa Cruz, sc-15407), active β-catenin (1:100, Millipore, 05-665), Vimentin (1:100, Abcam), Slug (1:100, Cell Signaling, 9585), Twist (1:100, Santa Cruz, sc-15393), Vimentin (1:100, Abcam, ab24525), and WT1 (1:100, Santa Cruz, sc-192) for 3 h at room temperature. After washing with secondary antibodies and Alexa Fluor 594 conjugated phalloidin (1:40, Molecular Probes) and Hoescht (Invitrogen), cultures were incubated for 1 h at room temperature. The slides were mounted with an anti-fade reagent (SlowFade gold, Invitrogen) and taken by an inverted microscope (Zeiss Axiovert 200 M) with an X40 oil immersion objective (Zeiss, 1.6 NA).

The fluorescent glucose analogue 2-NBDG (Molecular Probes, Thermofisher) was used for glucose uptake experiments. Cells were incubated in HBSS containing no glucose in the presence of 2 mM 2-NBDG for 25 min. Cells were then washed and 4–5 z-stacks per well were acquired using a Zeiss 710 VIS CLMS confocal microscope equipped with a META detection system and an x40 oil immersion objective (Zeiss, Oberkochen, Germany). The 488 nm laser was used to excite 2-NBDG and emitted fluorescence was measured above 495 nm. Green fluorescence inside the cells was quantified using Zeiss software. At least 90 cells per line were analysed in a minimum of 5 independent measurements.