Fig. 3: Neuronal C/EBPβ acts as a transcription activator for Dnm1l gene.

a Binding score of transcription factors bound to the Dnm1l promoter region determined by ChIP assays (ChIP-Atlas MACS2) in HeLa S3 cells obtained from the Signaling Pathway Project. b Measurement of mRNA levels of Gabpa, Ctcf, Rest, Cebpb in the control and susceptible mice. Data are presented the mean ± SEM (n = 6 for each group; two-sided unpaired t-test with Welch’s correction). c Correlation analysis between social interaction ratio and relative Cebpb mRNA levels in WT mice exposed to AcSD or control. The white, red, and blue dots represent the control, susceptible, and resilient groups, respectively. The Spearman correlation coefficient r² and two-sided p value are shown (n = 6 for each group). d Representative immunoblots of p-C/EBPβ and C/EBPβ protein levels in the mPFC of WT mice exposed to AcSD or control. Data are representative of three independent experiments. e The immunostaining of p-C/EBPβ (Red) with Dnm1l mRNA (Green) in situ hybridization on the mPFC sections of WT mice exposed to AcSD or control. Scale bar: 10 μm. Correlation analysis between relative p-C/EBPβ intensity and social interaction ratio (f), or relative Dnm1l mRNA intensity (g) in WT mice exposed to AcSD or control. The white, red, and blue dots represent the control, susceptible, and resilient groups, respectively. The Spearman correlation coefficient r² and two-sided p value are shown (n = 8 for each group). h Immunofluorescence co-staining of C/EBPβ (green) and DRP1 (Red) on the brain sections of the mPFC in WT mice exposed to AcSD or control. Scale bars: 20 μm. i Quantification of C/EBPβ intensity in the mPFC from WT mice exposed to AcSD or control. Data are presented the mean ± SEM (n = 8 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). j Correlation analysis between relative DRP1 intensity and relative C/EBPβ intensity in WT mice exposed to AcSD or control. The white, red, and blue dots represent the control, susceptible, and resilient groups, respectively. The Spearman correlation coefficient r² and two-sided p value are shown (n = 8 for each group). k Snapshots of C/EBPβ ChIP-Seq at locus of Dnm1l gene in HeLa cells, HepG2 cells, HCT116 cells and MSCs cells. l Luciferase assays with different truncates of Dnm1l promoter in HEK293 cells co-transfected with GST or GST-C/EBPβ. Data are presented the mean ± SEM (n = 6 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). m Representative immunoblots of C/EBPβ protein expressions in HEK293 cells co-transfected with variants lengths of Dnm1l promoter and GST-C/EBPβ. Data are representative of three independent experiments. n Luciferase assays with different truncates of Dnm1l promoter and representative immunoblotting bands of C/EBPβ protein expressions in HEK293 cells co-transfected with different truncates of Dnm1l promoter and si-control or si-C/EBPβ. Data are representative of three independent experiments. Data are presented the mean ± SEM (n = 5 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). o The consensus C/EBPβ-binding motif and sequence comparison of human and mouse Dnm1l promoters. p EMSA assay demonstrates that C/EBPβ binds the Dnm1l promoter. Nuclear extract proteins (NE) are isolated from HEK293 cells transfected with GST-C/EBPβ for 48 h. EMSA assay is recruited to detect the C/EBPβ binding ability on site –1369 to –1060 of Dnm1l promoter with probe (GTGGCGCAATC), mutation probe (GTGTATCAATC). Data are representative of three independent experiments. Probe bands indicate probe without binding of C/EBPβ protein. Shift bands indicate the C/EBPβ protein—Probe complex. Super-shift band indicates the C/EBPβ antibody—C/EBPβ protein—Probe complex. q ChIP-PCR assays demonstrate C/EBPβ specifically binds to Dnm1l promoter binding motif of genomic DNA. The C/EBPβ—DNA crosslinking ChIP samples are obtained from HEK293 cells and immunoprecipitated with anti-C/EBPβ or IgG antibodies. After reversing crosslinks, PCR are performed by using primer pairs at –1404 to –1195 of Dnm1l promoter. PCR assay also include each input sample. The positive control is demonstrated with anti-Histone H3 antibody coupling with GAPDH primers. Data are representative of three independent experiments. r, s Representative immunoblots and quantification of human and mouse C/EBPβ and DRP1 protein expressions in primary mouse neurons. Data are representative of three independent experiments. Data are presented the mean ± SEM (n = 5 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). t Measurement of Dnm1l mRNA levels in primary mouse neurons. Data are presented the mean ± SEM (n = 6 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). u Immunofluorescence co-staining of C/EBPβ (green) and DRP1 (Red) in primary mouse neurons. Scale bars: 10 μm. Quantification of C/EBPβ intensity (v) and DRP1 intensity (w) in primary mouse neurons. Data in (v) are presented the mean ± SEM (n = 10 for each group; ND not detected; two-sided unpaired t-test with Welch’s correction). Data in (w) are presented the mean ± SEM (n = 10 for each group; one-way ANOVA and Bonferroni’s multiple comparison test). See also Supplementary Fig. 6. Source data are provided as a Source Data file.