Figure 7: BRD4 inhibition inhibits bone cells differentiation.

(a,b) Purified human CD14⁺ monocytes were cultured for 13 days in the presence of M-CSF, RANKL and JQ1. TRAP colouration was performed at the end of the culture period (a) and multinucleated TRAP-positive cells were counted under a light microscope (scale bar, 100 μm) (b), t-test statistical analysis has been used. (c) Activity of the transcription factor NFkB as assessed with a luciferase report assay. HEK293-RANK were transfected with NFkB-Luc vector and then treated with and JQ1 (500 nM) for 24 h. Results show relative luciferase units (RLU, in %) normalized to the condition without RANKL. (d–f) qRT–PCR for NFATC1 (d), MMP9 (e) and CTR (calcitonin receptor) (f) RNA levels in human CD14⁺ monocytes cultured in the presence of M-CSF, RANKL and 125 nM JQ1 after 3 days (d) or 11 days (e,f). Analysis of variance (ANOVA), Bonferroni multiple comparisons statistical test has been used. Results are expressed as fold increase compared with the condition without RANKL and JQ1. (g) ChIP with a BRD4 antibody at two sites around the NFATC1 promoter region in cells treated with 500 nM JQ1 for 4 h and RANKL (100 ng ml⁻¹) for 1 h. Enrichment is shown as the percentage of total input DNA. The top track shows the levels of enrichment of the H3K27Ac histone mark across the genome as determined by a ChIP-seq assay on seven cell lines from ENCODE. (h–j) Human MSCs were cultured in OB differentiation medium (Diff. medium) and treated with JQ1. After 21 days, mineralization was quantified after staining with alizarin red-S (h). Expression of RUNX2 (i) and ALP (alkaline phosphatase) (j) RNA levels were assessed by qRT–PCR. Results are expressed as fold increase compared with the control condition without differentiation medium. ANOVA, Bonferroni multiple comparisons statistical test has been used. Error bars show s.e.m. for n=3 measurement from representative experiments.