Fv1000 software package

Intact neurons were loading with Fluo-4/AM or X-Rhod-1 AM (Life Technology) in transfected neurons, in HEPPS buffer for 30 min, and washed 3 times before imaging. Single isolated nuclei were loaded as reported^{24 (link)}. The membrane impermeant Ca²⁺ probe Fluo-4/dextran (30 μg/ml; 30 min at 4°C; Life Technologies) loaded the nucleoplasm while the membrane permeant Ca²⁺ probe Fluo-4/AM (20 μM; 60 min at 4°C) loaded the nuclear envelope. After loading, the nuclei were washed twice with the intracellular medium (125 mM KCl/2 mM K₂HPO₄/40 mM Hepes/0.1 mM MgCl₂, pH 7.2/100 nM Ca²⁺, with 10.2 mM EGTA and 1.65 mM CaCl₂) and then were equilibrated in the same medium supplemented with 1 μM of ATP and 300 nM Ca²⁺ for a few minutes to load nuclei with Ca²⁺. After that, they were washed twice again with the intracellular buffer (without ATP and Ca²⁺). Experiments were done at RT. No probe leakage was detected during the experiment. Ca²⁺ imaging in single isolated nuclei was performed by using an Olympus FV1000 Confocal microscope. Images were collected at 1 s intervals, and fluorescence was measured by using the Olympus FV1000 software package. The difference (ΔF) between the mean fluorescence measured in a given region of interest (ROI) and the corresponding control value for each ROI (F ₀) was expressed as fraction of the control (ΔF/F ₀) and was plotted as a function of time.

Isolated nuclei were equilibrated in intracellular medium with DiOC₆(3) (200 nM, RT, Life Technologies), which is a fluorescent cationic lipophyllic dye whose incorporation into lumen is proportional to ΔΨ^{24 (link)}. After 10 min, the nuclear envelope became stained. The nuclear were washed for 3 times before imaging with Olympus FV1000 Confocal microscope. Images were collected at 1 s intervals, and fluorescence was measured by using the Olympus FV1000 software package. The difference (ΔF) between the mean fluorescence measured in a given region of interest (ROI) and the corresponding control value for each ROI (F ₀) was expressed as fraction of the control (ΔF/F ₀) and was plotted as a function of time.