Fig 2: (a-d) 3T3-L1 fibroblasts were treated with differentiation medium for the indicated times (h, unless indicated by D = days) at which point samples were analysed for expression of Cebpb, Cebpd, Cebpa and Pparg by RT-qPCR. (e,f) 3T3-L1 fibroblasts were treated with the indicated components of the differentiation medium or the entire cocktail, as indicated, for 3 h at which point samples were analysed for expression of Cebpb and Cebpd by RT-qPCR. Data presented are mean ± SEM (n = 3)

Fig 3: CUL4B epigenetically represses Klf4 and Cebpd transcription. a, b qChIP assays were conducted to determine the binding regions for CUL4B at the promoters of Klf4 (a) and Cebpd (b) in BMSCs. The results are represented as the fold change over the control (IgG). c ChIP assays using BMSCs showed that CUL4B, DDB1, EZH2, H2AK119ub1, H3K27me3, HDAC1 and HDAC3 were enriched at the promoters of Klf4 and Cebpd. d, e qChIP assays of the recruitment of the indicated proteins at the Klf4 (d) and Cebpd (e) promoters in MKO and WT BMSCs (n = 3). H2A and H3 were used as controls. f, g Expression of Klf4 and Cebpd in CUL4B-overexpressing BMSCs (n = 3) treated with TSA and/or DZNep was detected by real-time qPCR (f) and Western blotting (g). h ChIP assays using ST2 cells showed that CUL4B, DDB1, EZH2, H2AK119ub1, H3K27me3, HDAC1 and HDAC3 were enriched at the promoters of Klf4 and Cebpd. i, j qChIP assays of the recruitment of the indicated proteins at the Klf4 (i) and Cebpd (j) promoters in control and Cul4b knockdown ST2 cells (n = 3). k, l Expression of Klf4 and Cebpd in CUL4B-overexpressing ST2 cells (n = 3) treated with TSA and/or DZNep was detected by real-time qPCR (k) and Western blotting (l). Error bars represent standard errors. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.000 1; ns, no significance

Fig 4: CUL4B negatively regulates Klf4 and Cebpd expression. a Heatmap of differentially expressed genes (DEGs) between primary cultured BMSCs from MKO and WT mice. Gene expression values are colored from blue (downregulated) to red (upregulated). b, c The expression of downregulated (b) and upregulated (c) DEGs in MKO BMSCs (n = 3) was verified by real-time qPCR. d The protein levels of KLF4 and C/EBPd in MKO and WT BMSCs were detected by Western blotting. e The mRNA levels of Klf4 in MKO and WT BMSCs (n = 3) during osteogenic differentiation were analyzed by real-time qPCR. f The mRNA levels of Cebpd in MKO and WT BMSCs (N = 3) during adipogenic differentiation were analyzed by real-time qPCR. g The mRNA and protein levels of KLF4 and C/EBPd in CUL4B-overexpressing and control BMSCs (n = 3) were analyzed by real-time qPCR and Western blotting. h The mRNA levels of Klf4 in CUL4B-overexpressing and control BMSCs (n = 3) during osteogenic differentiation were analyzed by real-time qPCR. i The mRNA levels of Cebpd in CUL4B-overexpressing and control BMSCs (n = 3) during adipogenic differentiation were analyzed by real-time qPCR. Error bars represent standard errors. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.000 1; ns, no significance