Te200 inverted fluorescent microscope

When E10 overexpressing TAZ or its mutants or A549 cells with or without TAZ/YAP knockout reached around 80% confluence, they were dissociated by trypsin-EDTA into single-cell suspensions. Triplicate of 1 × 10⁴ cells were suspended in Dulbecco's Modified Eagle Medium/F12 (Sigma-Aldrich Co. LLC., #D6421) supplied with L-glutamine, Epidermal Growth Factor (EGF, 20 ng/ml, Sigma-Aldrich Co. LLC., #E5036), Basic Fibroblast Growth Factor (bFGF, 20 ng/ml, Recombination human protein, AA10-155, Life Technology), and Insulin (4 μg/ml; Life Technologies, #A11429IJ). Cells were then seeded into each well of ultra-low-attachment 6-well plates (Corning, Inc., NY, USA). Following 7 days in culture, spheres that larger than 100 μm were quantitated and pictures were taken using TE200 Nikon Inverted Fluorescent Microscope (Nikon, Montreal, Canada). The diameter of each sphere is measured and average of diameter for each samples is regarded as sphere size.

Formalin fixed paraffin embedded (FFPE) tumor tissues were sectioned at the thickness of 3–4 μm, stained by the Discovery XT Automated IHC/ISH research slide staining system (Ventana Medical Systems, Inc.). Antigens were retrieved with EDTA (pH = 8) solution, blocked by 1% Bovine Serum Albumin (BSA, Fraction V), and incubated with mouse monoclonal anti-TAZ antibody (1:300 dilution, BD Biosciences) and rabbit monoclonal anti-ALDH1A1 (1:50 dilution, Abcam) antibody. As a control for specificity, one slide was processed with the same IHC condition except that primary antibody was not added. IHC signals were developed by using biotinylated HRP-conjugated anti-mouse or anti-rabbit secondary antibody, respectively, followed by catalyzing 3,3′-diaminobenzidine (DAB) substrate-chromogen into a visible precipitate. Pictures were taken using TE200 Nikon Inverted Fluorescent Microscope at 40× magnification.