Tubeness analysis, endogenous tubulin controls, and nuclear health controls. (A and B) Representative micrographs of COS-7 cells expressing (A) EGFP-DCX and (B) mCherry-α-tubulin before treatment (“baseline,” left), after 5-min treatment with 20 µM ZnCl₂ and 2.5 µM PTO (center), or after subsequent treatment with 100 µM TPEN (right). Scale bar = 10 µm. Raw fluorescence images (top) and “Tubeness” analysis (Fiji Plugin; bottom) are shown. (C) Representative micrographs of methanol-fixed COS-7 cells treated with 2 µM pyrithione in the presence (top) or absence (bottom) of 20 µM ZnCl₂. Fixed cells were incubated with anti-β-tubulin monoclonal antibodies (magenta, left), DAPI (cyan, center), and channels are shown merged (right). Scale bar = 10 µm. (D) Representative micrographs of COS-7 cells expressing LAMP1-mCherry (left), stained with Hoechst (middle), and merged images (right) before treatment (“baseline,” top), after 5-min treatment with 20 µM ZnCl₂ and 2.5 µM PTO (middle), or after subsequent treatment with 100 µM TPEN (bottom). Scale bar = 30 µm. (E) Representative micrographs of primary hippocampal neurons expressing mito-mCherry (left) and stained with Hoechst (middle) and merged images (right) before treatment (“baseline,” top), after 5-min Zn²⁺ influx by high K⁺ depolarization (middle), or after subsequent treatment with 100 µM TPEN (bottom). Scale bar = 30 µm. All experiments were performed in the absence of extracellular Ca²⁺.