Fig 2: Characterization of P19 cells used in the study. P19 cells were grown in differentiation media containing 1 µM retinoic acid (RA). Images were taken using an Evos microscope after 4 days of aggregation (A), and after P19 cells had formed neurons after differentiation treatment for 4 days (B). To further validate the neuronal differentiation, immunoblot (E) analysis was performed to show expression levels of the stem cell marker SSEA1, and the neuron-specific marker ß-3-tubulin in control (untreated) and differentiated (treated with 1 µM RA) P19 cells. ß-actin was used as a control or housekeeping protein, and was run on a separate gel, as shown in the supplementary information. SSEA1 and ß-3-tubulin were run together in another gel, as shown in the supplementary information. Immunostaining with SSAE1 was also performed (C) and (D). The red color is SSEA1 staining (C), and the blue color in both (C) and (D) shows nuclear Hoechst staining. The gene expression levels of Hes5, Neurod1 and Tubb3 were analyzed in untreated and differentiated P19 cells (F). Results show that the expression profile of the differentiated cells is visibly higher than that of the control.

Fig 3: Influence of SETDB1 knockdown on gene transcription and on the binding of EZH2 to promoters and the change in H3K9me3 and H3K27me3 on promoters.A, RT-qPCR detection of transcription of genes promoting neuronal differentiation, cell cycle, and growth arrest in control and knockdown HCT116 cells. Significance in transcription change was calculated based on experiments in triplicate using unpaired Student’s t test. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. NS, not significant. B, ChIP detection of binding of EZH2 to promoters of CDKN1A, GADD45B, BCL6, NEUROD1, TUBB3, and change in H3K9me3 and H3K27me3 in these promoters in response to knockdown of SETDB1 in HCT116 cells. Chromatin fragments were precipitated with antibodies against EZH2, H3K9me3, and H3K27me3, respectively, and detected with qPCR using primer pairs amplifying different regions of promoters (Table S7). Significance in difference in amplified promoter fragments was calculated using unpaired Student’s t test based on experiments in triplicate. Data are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ChIP, chromatin immunoprecipitation; NS, not significant; RT-qPCR, Reverse transcriptase-quantitative PCR.

Fig 4: Neuronal differentiation of primNSCs induced by RA treatment and by knockdown of Setdb1.A and B, neuronal differentiation phenotype in ESC-derived primNSCs that were treated with RA at an indicated dose and cultured in NSC-specific serum-free medium for a period as indicated (A), and IB detection of protein expression in control (DMSO) and treated (RA) cells (B). C and D, neuronal differentiation phenotype in ESC-derived primNSCs induced by knockdown of Setdb1 and cultured in serum-free medium (C), and IF detection of protein expression in control (shCtrl) and knockdown (shSetdb1) cells (D). E–G, neuronal differentiation phenotype in NE-4C cells induced by knockdown of Setdb1 and cultured in serum-free medium (E), and IF (F) and IB (G) detection of protein expression in control (shCtrl) and knockdown (shSetdb1) cells. H and I, enrichment analysis on GO and KEGG pathway terms for upregulated (H) and downregulated (I) genes identified in a transcriptome profiling on control and knockdown cells in (E). In (D) and (F), cell nuclei were counterstained with DAPI. In (F), Map2 and Tubb3 were simultaneously detected with cells on the same coverslips, either control (shCtrl) or knockdown cells (shSetdb1). ESCs, embryonic stem cells; IF, immunofluorescence; IB, immunoblotting; NSCs, neural stem cells; primNSCs, primitive NSCs; RA, retinoic acid.

Fig 5: Adipose RBM3 mediates the action of S100A8+ immune cells in sympathetic innervation.a Schematic diagram of experimental processes. This diagram was created with BioRender.com. b Representative images of TH staining in the BAT of mice injected with AAV-RBM3 or AAV-NC and transferred with S100A8+ immune cells. Scale bar, 100 µm. c Representative immunoblots of UCP1, TH, p-TH, TUBB3 and RBM3 in the BAT of mice injected with AAV-RBM3 or AAV-NC and transferred with S100A8+ immune cells (n = 3). d Relative mRNA levels of Rbm3, Th Ucp1, Ppargc1a, p16, and p21 in the BAT of mice injected with AAV-RBM3 or AAV-NC and transferred with S100A8+ immune cells (n = 5). e Oxygen consumption of mice injected with AAV-RBM3 or AAV-NC and transferred with S100A8+ immune cells or S100A8- immune cells (n = 6–7). f Core body temperature of mice injected with AAV-RBM3 or AAV-NC were transferred with S100A8+ immune cells and subjected to cold challenge (n = 5). g Venn diagram showing overlapped genes which are RBM3-bounded and differentially expressed upon Rbm3 knockdown. h KEGG analysis of the 909 overlapped genes shown in (g). i Heatmap of axon guidance-related gene expressions based on RNA-seq data of Rbm3 knockdown differentiated brown adipocytes. j Relative mRNA levels of Rbm3 and axon guidance-related genes Nrp1 and Epha7 in differentiated brown adipocytes transfected with si-Rbm3 or si-NC (n = 3). k Representative immunoblots of NRP1 and EPHA7 in differentiated brown adipocytes transfected with si-Rbm3 or si-NC (n = 4). l RNA immunoprecipitation (RIP) assay assessing RBM3 binding on 3´UTR of Nrp1 and Epha7 in differentiated brown adipocytes (n = 4). m Relative mRNA level of Nrp1 and Epha7 in si-Rbm3 or si-NC transfected brown adipocytes upon transcriptional inhibition with actinomycin D at indicated time (n = 3). n Representative images of TUBB3 staining in PC12 cells cocultured with differentiated brown adipocytes overexpressed with RBM3 and transfected with si-Nrp1 or si-Epha7 (n = 4). Scale bar, 100 µm. Data shown are representative of three independent experiments with similar results. n indicates the number of biologically independent samples examined. Data are shown as the mean ± SEM. Statistical differences were supposed to be significant when P < 0.05. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple-comparison test (n) or two-way ANOVA (f, m), ANCOVA with body weight as covariant (e) or unpaired two-tailed Student’s t test (c, d, j–l). Source data are provided as a Source Data File.