Fig. 4.

BCI inhibits differentiation of BMCs into macrophages. Bone-marrow cells (BMCs) were isolated and cultured from the femurs and tibias of 6-week-old wild-type Sprague-Dawley rats. (A) Bright-field images of BMC-derived macrophages, and the numbers of adherent macrophages in each HPF after 7-day induction by M-CSF in the presence of DMSO or BCI (scale bar: 100 μm; n=3 per group). (B) Numbers of adherent macrophages in each HPF at different time points of BCI treatment (n=3 per group). (C) Expression levels of Egr1, Grb2 and Mnda mRNAs in BMCs at day 3 after M-CSF induction with DMSO or BCI treatment (n=3 per group). (D) Western blots showing DUSP6 expression in M-CSF-induced bone-marrow-derived macrophages (BMDMs) from wild-type (WT) or Dusp6 mutant rats (n=3 per group). (E) Bright-field images of BMC-derived macrophages and the numbers of adherent macrophages in each HPF after 7-day induction by M-CSF from WT or Dusp6 mutant rats (scale bar: 100 μm; n=3 per group). (F) Expression levels of Egr1, Grb2 and Mnda mRNAs in BMDMs at day 3 after M-CSF induction from WT or Dusp6 mutant rats (n=3 per group). (G) RNA-seq showing differential gene expression in myeloid cells at 3 days after M-CSF induction with or without BCI. (H) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of genes downregulated by BCI revealed the top 10 affected pathways. (I) Gene Ontology (GO) analysis of genes downregulated by BCI showed ten signaling pathways including cytokines/chemokines. (J) Heatmaps showing the downregulation of cell adhesion molecules, chemokine signaling pathway, growth factor receptor binding and osteoclast differentiation after BCI treatment. One-way ANOVA followed by Dunnett's multiple comparison test; mean±s.e.m.; *P<0.05, **P<0.01, ***P<0.001.