Immunofluorescent staining was performed based on the methods described previously (Mo et al. 2015 (link); Yuan et al. 2021 (link)). Briefly, cells were seeded into 4-well LAB-TEK® II chamber slides (Nalge Nunc International, IL). After exposure to Nano-CuO for 48 h, cells were fixed with 10% neutral buffered formalin for 10 min and rinsed with 1x PBS three times (5 min each). Then cells were then incubated in a blocking solution (3.26% of BSA, 5% of normal goat serum, and 0.3% Triton X-100) for 1 h at room temperature for cell permeabilization and blocking of the nonspecific protein binding. After incubation with anti-E-cadherin (1:200) and anti-vimentin (1:500) antibodies overnight at 4 °C, the cells were incubated with Alexa Fluor® 488-conjugated goat anti-rabbit IgG and Alexa Fluor® 594-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA) for 1 h at room temperature. The slides were mounted with Prolong Gold Antifade Reagent with DAPI (Invitrogen) and images were captured under fluorescence microscopy (Nikon, Japan).