Ckx53 inverted

All images were taken with either an Olympus IX83 or CKX53 inverted microscope (Olympus, Tokyo, Japan) and analyzed by ImageJ. Fluorescence images of the permeability study were obtained using a FV‐1000 confocal laser scanning microscope (Olympus). The permeability was quantified by the mean fluorescence of the MCTS.

A series of MCF-10A cells (2 x 10^4 per well) were mixed with 0.3% agarose, low gelling temperature (Cat#35640, SIGMA) in the full growth medium, plated on top of a solidified layer of 0.6% agarose in full growth medium [25 (link)] in a 6-well plate, and fed every 3 days with full growth medium. Photographs were taken by an OLYMPUS CKX53 inverted microscope with a DP20 digital camera (OLYMPUS). Finally, the colonies were stained with MTT (1 mg/ml in PBS solution) and imaged using an EPSON GT-X900 scanner. The images were analyzed with ImageJ (Fiji), extracting the number of colonies that exceeded a certain threshold intensity. When we combined soft agar assay with the siRNA experiment, 1 nM siRNA-treated cells were embedded in the soft agar, fed only one week later and cultured for 2 weeks. MCF-10A cell lines were transfected with 1 nM siPOOLs, maintained for 2 days in partial growth medium, and then 2 x 10^4 cells were embedded in full growth medium-based soft agar. In the case of cancer-derived cell lines, cells were first transfected with 1 nM siPOOLs in RPMI supplemented with 10% FBS. One day after transfection, the cells were embedded in the soft agar based on RPMI supplemented with 10% FBS. The cell number embedded in the soft agar is as follows: A549, 2 x 10^4 cells; H358, 4 x 10^4 cells; A375, 1 x 10^4 cells.

Brightfield images were taken using an Olympus CKX53 inverted microscope with a 4x objective (UPlanFL N 4x/0.13 NA) and acquired with the OLYMPUS cell Sens Standard v2.3 software. Fluorescent images were captured within a week from staining using a Leica DMI6000B widefield microscope or a Leica TCS SP8 laser scanning confocal microscope. The image acquisition software was Leica LAS X and images were processed using Adobe Photoshop CC 2020. In comparison experiments, all the images were taken with the same software settings and any adjustments were applied equally across the entire image, without the loss of any information.