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Figure 4. TAK1 inhibition suppresses osteoclastogenesis enhanced by RANKL and multiple myeloma cells. (A) RAW264.7 cells were starved in α-MEM with 1% fetal bovine serum (FBS) for 12 hours, and then treated with or without LLZ at 1 mM for 3 hours, followed by the addition of RANKL (20 ng/mL) for the indicated time peri- ods. Phosphorylated TAK1 (p-TAK1), TAK1, IκBα, phosphorylated p38MAPK (p-p38MAPK), p38MAPK, phosphorylated ERK (p-ERK), and ERK protein levels were ana- lyzed using western blotting. b-actin was used as a loading control. (B) In order to observe p65 localization, the cells were fixed and stained with anti-p65 antibody (red), and their nuclei were stained with Hoechst 33342 (blue). (C) RAW264.7 cells were treated with or without RANKL at 25 ng/mL for 1 day. LLZ was added as indicated. Protein levels of NFATc1 and c-fos were determined using western blotting. (D) Primary mouse bone marrow cells were cultured in quadruplicate with or without RANKL (25 ng/mL) for 4 days. LLZ was added at the indicated concentrations. Then, the cells were fixed, and stained by TRAP. TRAP-positive multinucleated cells containing three or more nuclei were counted (left). Data are expressed as mean ± standard deviation (SD). *P<0.05 (the difference from the results with RANKL without LLZ). Microscopic images of TRAP staining in representative cultures are shown (right). Original magnification, x100. (E) RAW264.7 cells were transfected with scrambled (siCTL) or TAK1 (siTAK1) small interfering RNA, and then cultured with or without RANKL (25 ng/mL) for 24 hours. Cell lysates were then collected, and TAK1, NFATc1 and c-fos protein levels were assayed by western blotting. b-actin served as a loading control. (F) RAW264.7 cells transfected with scrambled (siCTL) or TAK1 (siTAK1) were cultured in quadruplicate with or without RANKL (25 ng/mL) for 4 days. TRAP-positive multinucleated cells containing three or more nuclei were counted. Data were expressed as mean ± standard devaition (SD). *P<0.05. (G) Primary bone marrow cells derived from mice were cocultured in quad- ruplicate with MM cells as indicated for 4 days with or without LLZ. The cells were then fixed, and stained by TRAP. TRAP-positive multinucleated cells containing three or more nuclei were counted. Data were expressed as mean ± SD. *P<0.05.
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haematologica | 2021; 106(5)