Fig. 5. Abnormal transient transcriptional profile in hippocampus of Tob-KO mice and suppressed stress-induced LCN2 expression induced after fear conditioning training.

A Heatmaps for differentially expressed genes in hippocampi of Tob-KO compared to Tob-WT mice using RNA sequencing without fear conditioning training (naive) and 15 min, 1 h, 3 h after fear conditioning training (represented as z-scores of log raw counts, FC_upregulated > 2 FC_{downregulated} < 0.5, p < 0.05, FDR < 0.05). B Pathway analysis for RNA sequencing candidates using IPA software showing activation of hormonal concentration in hippocampus of Tob KO mice at 15 min post-conditioning. C Real-time PCR for lipocalin-2 (Lcn2) mRNA in hippocampus of Tob-WT and KO naive mice and 15 min, 1 h and 3 h after fear conditioning. Two-way ANOVA followed by Bonferoni’s post-hoc test for multiple comparisons. D Western blotting showing protein expression of LCN-2 in hippocampi of naive Tob-KO mice and at 15 min, 1 h, and 3 h after fear conditioning training. E Normalized band intensity for LCN2 protein immunoblots. Two-way ANOVA followed by Bonferoni’s post-hoc test for multiple comparisons. F Western blotting showing abnormal protein expression in hippocampi of mice lacking Tob before and after fear conditioning training at 15 min, 1 h and 3 h. G Western blot band intensity quantification plots at different time points post-training compared to naive Tob-WT (p-ERK n = 4, MKP-1 n = 3). All values represent means ± SEMs. ns non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.