Fig 1: Foot shock strength dependently induces fear memory generalization with TET3 expression in NAc and knockdown of TET3 in NAc with TET1 and TET2 unaffected. (A) Experimental protocol. Day 1, tone-habituation and conditioning. Day 2, testing. CS, 10 s; US, 1 s; average intertrial interval () = 70 s (40–100 s). (B) The histogram represents freezing % to CS- and CS+ in no shock group (n = 11), weak shock group (n = 13), and strong shock group (n = 13). (C) The histogram represents the index behavior of generalization (IBG) of the three groups, IBG was calculated by the ratio of the freezing % of CS- to CS+ (no shock group: N = 11, weak shock group: N = 13, strong shock group: N = 13). (D and D') Western blotting shows the levels of TET1, TET2, TET3, and TDG in NAc of the control group, weak shock group, and strong shock group. Data are standardized to the control group (n = 4, from 8 mice, each sample in triplicate). (E) Immunofluorescence micrographs of the AAV virus injection site. Scale bars = 200 µm. Immunofluorescence micrographs of colabeling of GFP + cells and Neun+ neurons. Scale bars = 100 µm (left), 50 µm (right). (E') The number of GFP+ - NeuN + cells and GFP+ cells. The percentage of colabeling cells is about 95.38% (n = 6 images per mice, n = 6). (F) Immunofluorescence micrographs of CTR (n = 5) and shTet3 group (n = 6). Scale bars = 100 µm (left), 50 µm (right). Six micrographs for each mouse. DAPI, 4',6-diamidino-2-phenylindole. (F') Quantification of GFP+ cells in NAc from CTR mice and shTet3 mice. (F?) Quantification of the TET3+ cell average optical density in NAc. (G and G') Western blotting shows the levels of TET3, TET1, and TET2 expression in CTR and shTet3 group (n = 4, from eight mice, each sample in triplicate). (H) Quantitative PCR for mRNA expression of Tet1, Tet2, Dnmt3a, Dnmt3b, and Dnmt1. Data are standardized to the CTR group (n = 4, from 12 mice, each sample in triplicate). Quantifications are presented as means ± SEM. (B) Two-way ANOVA with Tukey's test; (C, F', F?, G, and H) Unpaired two-tailed t-test; (D') One-way ANOVA with Tukey's test; **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant