Journal: Experimental and Therapeutic Medicine
Article Title: 4-Hexylresorcinol inhibits osteoclastogenesis by suppressing the NF-κB signaling pathway and reverses bone loss in ovariectomized mice
doi: 10.3892/etm.2021.9785
Figure Lengend Snippet: 4HR suppresses RANKL-induced activation of the NF-κB pathway. (A-E) BMMs cultured at 37˚C with complete medium supplemented with 10 ng/ml M-CSF and 100 ng/ml RANKL. (A) Representative images of western blot analysis showing that 4HR inhibited the RANKL-induced protein expression of TRAP, NFATc1 and Cathepsin K in BMMs after 20 µg/ml 4HR treatment for 3 days in the presence of 10 ng/ml M-CSF and 100 ng/ml RANKL. (B) Quantification of (A). * P<0.05, ** P<0.01, *** P<0.001 vs. Veh. (C) Luciferase assay of the NF-κB luciferase reporter plasmid in BMMs pretreated with or without 4HR (20 µg/ml) for 30 min in the presence or absence of 10 ng/ml M-CSF and 100 ng/ml RANKL. *** P<0.001 vs. Veh without 4HR treatment and RANKL stimulation, ### P<0.001 vs. BMMs without 4HR treatment but with RANKL stimulation. (D) Western blot analysis of the lysate following 100 ng/ml RANKL incubation for the indicated time points (0, 15, 30 and 60 min) in BMMs with or without 4HR preincubation for 30 min (20 µg/ml) in the presence of 10 ng/ml M-CSF. (E) Quantification of (D). * P<0.05, ** P<0.01 and *** P<0.001 vs. Veh without 4HR exposure at the corresponding time points. 4HR, 4-hexylresorcinol; RANKL, receptor activator of NF-κB ligand; TRAP, tartrate resistant acid phosphatase; NFATc1, nuclear factor of activated T-cells cytoplasmic 1; BMMs, bone marrow-derived macrophages; M-CSF, macrophage colony-stimulating factor; Veh, Vehicle; p-IKKβ, phosphorylated-IκB kinase β.
Article Snippet: Rabbit polyclonal anti-TRAP (cat. no. 11594-1-AP; 1:1,000), Mouse monoclonal anti-nuclear factor of activated T-cells cytoplasmic 1 (NFATc1; cat. no. 66963-1-Ig; 1:1,000) and rabbit polyclonal anti-Cathepsin K (cat. no. 11239-1-AP; 1:1,000) antibodies were from ProteinTech Group, Inc. Rabbit monoclonal anti-phosphorylated (p)-IκB kinase β (pIKKβ; cat. no. 2697S, 1:1,000) and total anti-IKKβ rabbit monoclonal (cat. no. 8943S; cat. no. 1:1,000) antibodies were obtained from Cell Signaling Technology, Inc. Recombinant soluble M-CSF and RANKL were obtained from PeproTech, Inc.
Techniques: Activation Assay, Cell Culture, Western Blot, Expressing, Luciferase, Plasmid Preparation, Incubation, Derivative Assay
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