Figure 1. VIVIT-GFP overexpression counteracts the effects of constitutively activated calcineurin. A, Representative pictures of GFP, NFATc4 immunostaining, and DAPI (top row) in cortical primary neurons transfected with WtCaN or CACaN, altogether with either GFP or VIVIT-GFP (transfected cells are indicated by an arrow). An increased colocalization of the NFATc4 immunostaining and DAPI is detected in cells overexpressing CACaN and GFP (second row), which is abolished when VIVIT-GFP is cotransfected. The bottom row represents the detection of NFATc4 immunostaining in the nucleus and in the cytoplasm using ImageJ. Scale bar, 50 μm. B, NFATc4 ratios between the nucleus and the cytoplasm are significantly increased when CACaN is overexpressed compared with GFP and WtCaN+GFP. When CACaN is co-transfected with VIVIT-GFP, the nucleus/cytoplasm ratios are significantly decreased and become comparable to neurons transfected with WtCaN+GFP. C, Analysis of NFAT transcriptional activity using the Luciferase reporter system shows that overexpression of CACaN leads to a significant higher Luciferase signal (i.e., higher NFAT transcriptional activity) compared with WtCaN and CACaN+VIVIT. D, E, Representative images and spine density quantification of neurons transfected with GFP, WtCaN/GFP, CACaN/GFP, or CACaN/VIVIT-GFP show that cells co-transfected with CACaN and GFP have a decreased spine density compared with GFP or WtCaN+GFP overexpressing neurons, which is significantly increased by VIVIT-GFP. Scale bar, 100 μm. F, The proportion of each spine type is also affected by CACaN and a significant decrease of the more mature “mushroom” spines is observed compared with GFP and WtCaN/GFP transfected neurons. This effect is greatly abolished when VIVIT-GFP is overexpressed. G, The overall dendritic complexity is also improved when the VIVIT inhibitor was coexpressed with CACaN compared with CACaN/GFP transfected cells. No statistical difference is observed between GFP, WtCaN/GFP, and CACaN/VIVIT-GFP transfected cells. *p < 0.05, **p < 0.001 and ***p < 10−5. (n > 40 cells per condition).