Fig. 5

CHEK1 induces macrophage osteoclast by upregulating NFATc1 expression. A Magnetic resonance imaging (MRI) revealed that increased CHEK1 expression was positively correlated with bone lesion formation in TT2 cohort MM patients. B–C TRAP staining revealed that Chek1 OE promoted osteoclast differentiation in RAW 264.7 mouse macrophages co-treated with RANKL (50 ng/mL) and M-CSF (15 ng/mL) in a time-dependent manner. D–E TRAP staining confirmed that Chek1 OE prompted osteoclast differentiation in RAW 264.7 cells treated with varying doses of RANKL and M-CSF in a manner dependent on RANKL and M-CSF dosages. F–G TRAP staining revealed that human primary peripheral blood mononuclear cells (PBMCs) transfected with human CHEK1 cDNA developed significant more osteoclasts than non-transfected control cells. H–I Western blotting and TRAP staining confirmed that the CHEK1 inhibitor LY2603618 decreased NFATc1 expression and suppressed osteoclast differentiation in RAW 264.7 cells. J Co-IP revealed that CHEK1 interacted with NFATc1 in RAW 264.7 cells. K Western blotting confirmed that the expression of NFATc1 was increased in Chek1-OE RAW264.7 cells relative to WT cells. L CHEK1 knockdown prevented myeloma-associated bone loss in 5TMM3VT model. Micro-CT analysis of 5TMM3VT-involved tibia bone performed at 4 weeks confirmed the presence of osteolytic lesions and demonstrated decreased trabecular bone volume (BV/TV) compared with CHEK1 gene knockdown