Journal: JBMR Plus

Article Title: Osteocytes regulate osteoprotegerin expression via the p38-MAPK-CREB pathway in rheumatoid arthritis

doi: 10.1093/jbmrpl/ziag023

Figure Lengend Snippet: TNF-α-induced changes in Wnt/β-catenin-related transcripts and activation of the p38-MAPK-CREB signaling pathway in osteocytes. (A) Dot plots showing relative mRNA levels of Ctnnb1 (β-catenin), Lef1 , and Axin2 in MLO-Y4 cells treated with or without TNF-α ( n = 3). (B) Representative immunoblotting images showing phosphorylated p38 MAPK (p-p38), total p38, phosphorylated CREB (p-CREB), total CREB, phosphorylated JNK (p-JNK), total JNK, phosphorylated ERK (p-ERK), total ERK, and β-actin in MLO-Y4 cells following TNF-α treatment. Data in (A) are shown as individual values with the mean ± SD and were analyzed by the two-sided Student’s t -test. (C) Representative immunoblot of OPG in TNF-α-stimulated MLO-Y4 cells treated with a p38 inhibitor or a CREB inhibitor. Cells were stimulated with TNF-α in the presence or absence of SB203580 (p38 inhibitor) or 666-15 (CREB inhibitor), and total cell lysates were subjected to immunoblotting for OPG. β-actin serves as the loading control. Numeric values shown beneath each band represent the relative expression levels of OPG/β-actin for the corresponding lane. (D) ChIP-PCR demonstrating enrichment of the OPG promoter in anti-CREB immunoprecipitates from MLO-Y4 cells with or without TNF-α stimulation, with densitometric quantification of the ChIP-PCR bands; input DNA served as the internal control, normal IgG as the negative control, and anti-histone H3 as the positive control.

Article Snippet: Quantitative PCR was performed using TaqMan Gene Expression Assays (Thermo Fisher Scientific) for the following targets: Tnfsf11 (Mm00441906_m1), Tnfrsf11b (Mm00435454_m1), Sost (Mm00470479_m1), Ctnnb1 (Mm00483039_m1), Axin2 (Mm00443610_m1), Lef1 (Mm00550265_m1), and Acp5 (Mm00475-698_m1).

Techniques: Activation Assay, Western Blot, Control, Expressing, Negative Control, Positive Control