Fig 2: RA blocked GC-induced changes in the Ppp2ca-SUV39H1-p47phox pathway and stress-induced depression-like behavior in aged mice.a–d Ppp2ca, SUV39H1, and p47phox transcript levels in HT22 cells treated with GC GC + RA, or RA alone. RA, 50 or 100 µM (n = 6, each; a One-way ANOVA, F(2,15)=8.933, p = 0.0028; b One-way ANOVA, F(2,15)=27.62, p < 0.0001; c One-way ANOVA, F(2,15)=13.22, p = 0.0005; d One-way ANOVA, F(2,15)=12.11, p = 0.0007). e, f Photomicrographs showing DHE-reactive ROS levels in HT22 cells treated with GC, and GC plus RA, and quantification levels (n = 5, each; One-way ANOVA, F(2,12)=5.31, p = 0.0223). g Experimental design for treatment with daily 2-h restraint for 5 days (RST5d) or RST5d plus RA in aged (18M) mice, and following behavioral tests. Arrow, time point for tissue preparation. h–k Expression levels of Ppp2ca, SUV39H1, p47phox, and gp91phox transcripts in the hippocampus of indicated groups (n = 6, each; Ppp2ca, One-way ANOVA, F(2,15)=4.458, p = 0.0302; SUV39H1, One-way ANOVA, F(2,15)=15.42, p = 0.0002; p47phox, One-way ANOVA, F(2,15)=15.83, p = 0.0002; gp91phox, One-way ANOVA, F(2,15)=4.249, p = 0.0345). l Representative tracking plots and time spent in the target and non-target fields (n = 7 mice/group; target field, One-way ANOVA, F(2,18)=8.003, p = 0.0033; non-target field, One-way ANOVA, F(2,18)=7.969, p = 0.0033). m Sociability index in the sociability test (n = 7 mice/group; One-way ANOVA, F(2,18)=7.983, p = 0.0033). n Immobility time in the FST (n = 7 mice/group; One-way ANOVA, F(2,18)=13.22, p = 0.0033). o, p K-Means clustering of individuals in the sociability x FST matrix plotting with z-scores and % composition of each group in the clusters (cluster1, 100% for CON, 14.3% for RST5d, and 85.7% for RST5d + RA; cluster2, 0% for CON, 85.7% for RST5d, and 14.3% for RST5d + RA). *p < 0.05 and **p < 0.01. One-way ANOVA followed by a Newman–Keuls post hoc test.

Fig 3: SUV39H1 negatively regulated p47phox and gp91phox expression.a Experimental design. siSUV39H1 or siCON was stereotaxically injected in the CA3 region (red arrows). Blue arrow, tissue preparation point. b Expression levels of SUV39H1, p47phox, gp91phox, and p67phox transcripts in the CA3 region (n = 8, each). c Photomicrographs showing DHE-stained CA3 region of mice injected with siSUV39H1 or siCON. Scale bars, 200 µm. d Quantification of DHE-reactive ROS levels (n = 7 mice/group). e Diagram showing the promoter region of the p47phox. f–i ChIP-qPCR analysis showing the levels of SUV39H1, trimeH3K9, dimeH3K9, and acH3K9 binding to the promoter of the p47phox in the hippocampus of mice at 2 and 18 months of age. P1 and P2, the promoter regions used for ChIP-qPCR analysis. j Diagram showing the promoter region of the gp91phox. k–n ChIP-qPCR analysis showing the levels of SUV39H1, trimeH3K9, dimeH3K9, and acH3K9 binding to the promoter of the gp91phox in the hippocampus of mice at 2 and 18 months of age. P1 and P2, the promoter regions used for ChIP-qPCR analysis (p47phox, SUV39H1 n = 6, trimeH3K9, n = 5, dimeH3K9, n = 4, and acH3K9, n = 5; gp91phox, SUV39H1 n = 6, TrimeH3K9, n = 4, DimeH3K9, n = 7, and acH3K9, n = 4). *p < 0.05 and **p < 0.01.