Zinc Imaging and Cell Cycle Analysis

1 × 10⁵ cells were grown on 0.17 mm thick coverslips for 5–7 days prior to transfection. Coverslips were fixed and processed as previously described [13 (link)]. For zinc imaging, cells were loaded with 5 μM Fluozin-3 (Invitrogen) for 30 min at 37 °C. For FACS analysis using a Becton–Dickinson FACSVerse, non-adherent cells collected by mitotic shake-off and adherent cells harvested by trypsinisation were loaded with 5 μM Fluozin-3 (Invitrogen) for 30 min followed by 30 min recovery in medium. For cell cycle analysis, cells were fixed in 70% ethanol overnight followed by DNA staining with 20 µg/mL propidium iodide (Sigma-Aldrich) plus 0.2 µg/mL DNase − free RNase A and 0.1% Triton X-100 in PBS at 37 ºC for 20 min before FACS analysis and analysed with FlowJo Software version 10 using Watson pragmatic algorithm. Scale bar is 10 μm.