Fig 2: Requirement of NFATc4 in neuritin-mediated formation of dendritic spines in PFC neurons. A and B, representative images and statistical analyses of the effect of neuritin on PFC neuron spine density in C57BL/6 and Nfatc4-/-mice. Identification of neurons was performed using Golgi staining. C, effect of neuritin on spine length of PFC neurons in C57BL/6 or Nfatc4-/-mice. Cumulative frequency distributions of spine length were analyzed using a one-way Kruskal-Wallis test followed by Kolmogorov-Smirnov test. The median and quartiles of the control group and neuritin-treated group were compared. D, Sholl analysis of the branch intersection numbers. E and F, effect of neuritin on first and second order dendrite length of PFC neurons in C57BL/6 or Nfatc4-/- mice. ***, p < 0.001 compared with corresponding control by one-way ANOVA followed by Fisher's post hoc test.

Fig 3: Western blot analysis of endogenous NFATC4, STIM1, and PPP3R1 protein levels in miR-34a-5p-overexpressing Jurkat cells.a, b Representative western blot images. Jurkat cells were transfected for 48 h either with non-targeting control (ANC) or synthetic miR-34a-5p mimic. NFATC4, STIM1, and PPP3R1 protein was detected by specific monoclonal antibodies in western blot analysis. ß-Actin served as loading control. c–e Quantification of NFATC4, STIM1, and PPP3R1 expression by densitometric analysis of three independent western blot experiments. Respective protein expression was standardized according to ß-Actin loading control and expression level of the control transfected cells was set to 100%. Results are shown as means of five independent experiments with corresponding standard errors (SEM). Statistical evaluation was performed using Student’s t-test. A normal distribution of the data was assumed. (*p = 0.05, **p < 0.01, ***p < 0.001; PPP3R1 protein phosphatase 3 regulatory subunit B alpha, NFATC4 nuclear factor of activated T cells 4, STIM1 stromal interaction molecule 1

Fig 4: Lercanidipine inhibits Ang II-induced NFAT3 nuclear translocation. After 24 h serum starvation, NRVMs were treated with Ang II (2×10-7 M) for 24 h. FK506 (10-7 M) or lercanidipine (10-6 M) was added to the culture medium 1 h prior to Ang II administration. Control cells received no treatment. (A) Representative images of immunofluorescence staining of NFAT3. (B) Semi-quantitative analysis of NFAT3 nuclear translocation. Data are presented as the mean ± standard error of the mean. #P<0.05 vs. the control group; *P<0.05 vs. the Ang II group. Ang II, angiotensin II; Lercan, lercanidipine; NFAT3, nuclear factor of activated T cells 3; NRVMs, neonatal rat ventricular myocytes.

Fig 5: Lercanidipine inhibits Ang II-induced activation of Cn-NFAT3 and CaMKII-HDAC4 signaling in NRVMs. After 24 h serum starvation, NRVMs were treated with Ang II (2×10-7 M) for 24 h. FK506 (10-7 M) or lercanidipine (10-6 M) was added to the culture medium 1 h prior to Ang II administration. Control cells received no treatment. (A) Representative western blotting images. Densitometric analysis of (B) CnA, (C) NFAT3, (D) CaMKII and (E) HDAC4 expression levels. Data are presented as the mean ± standard error of the mean. #P<0.05 vs. the control group; *P<0.05 vs. the Ang II group. Ang II, angiotensin II; CaMKII, calcium/calmodulin-dependent kinase II; CnA, calcineurin A; HDAC4, histone deacetylase 4; Lercan, lercanidipine; NFAT3, nuclear factor of activated T cells 3; NRVMs, neonatal rat ventricular myocytes.