Bs 14 rc

HTR8/SVneo cells ( $4\times {10}^4$ per well) were seeded into the slides (BS-14-RC; Biosharp) for 20 h, and we allowed them to adhere for 20 h. After adhesion, cells were cultured with $2\mathrm{\mu }\mathrm{M}$ of ${\text{NaAsO}}_2$ for 24 h. Each group contained three biologically independent replications. Before immunofluorescence staining, slides were washed twice with phosphate-buffered saline (PBS). HTR8/SVneo cells were fixed with precold methanol for 5 min at room temperature. Subsequently, slides were incubated in blocking buffer [1% bis(trimethylsilyl)acetamide (BSA) in PBS] for 45 min and then with rabbit anti-CYR61 (ab228592, 1:600 dilution) overnight at 4°C. After being washed with PBS, the slides were incubated with Cy3-labeled goat anti-rabbit IgG (A0516; Beyotime; 1:400 dilution) for 60 min at room temperature. Slides were washed four times with PBS. The nucleus was counterstained with Hoechst 33342 (C1025; Beyotime; 1:400 dilution) for 5 min at room temperature. Slides were quickly washed three times in PBS and coverslipped with mounting medium. All slides were observed by fluorescence microscope (BX53F; Olympus).