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 most strongly modulates osteocyte-related gene and protein expression. (A) Dot plots of the relative mRNA levels for Tnfsf11 (RANKL) and Tnfrsf11b (OPG) in MLO-Y4 cells stimulated with TNF-α, IL-6 or IL-17 ( n = 3). (B) Dot plots showing enrichment of the osteocyte marker Sost in osteocyte-enriched bone fraction (OEBFs) vs non-digested bone ( n = 3). (C) Dot plots of Tnfsf11 , Tnfrsf11b , and Sost mRNA in OEBF with or without TNF-α ( n = 3). (D) Representative immunoblot of OPG and Sclerostin in OEBF with or without TNF-α; β-actin serves as the loading control. (E) Dot plots of secreted OPG in conditioned medium from MLO-Y4 cells treated with TNF-α, measured by ELISA ( n = 3). Data in (A-C and E) are individual values with the mean ± SD; * p < .05, ** p < .01, *** p < .001; ns indicates not significant by the 2-sided Student’s t -test.

Article Snippet: For osteoclast differentiation assays, the following 5 conditions were compared (all containing 100 ng/mL RANKL): (1) no MLO-Y4 CM and no TNF-α (RANKL only; no TNF source); (2) TNF-α added directly to osteoclast cultures (TNF-α, no MLO-Y4); (3) Unstim CM added at 10% (v/v); (4) TNF-stim CM added at 10% (v/v); and (5) TNF-stim CM pre-incubated with a neutralizing anti-OPG antibody (AF459, R&D Systems) prior to addition to osteoclast cultures.

Techniques: Expressing, Marker, Western Blot, Control, Enzyme-linked Immunosorbent Assay

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: For osteoclast differentiation assays, the following 5 conditions were compared (all containing 100 ng/mL RANKL): (1) no MLO-Y4 CM and no TNF-α (RANKL only; no TNF source); (2) TNF-α added directly to osteoclast cultures (TNF-α, no MLO-Y4); (3) Unstim CM added at 10% (v/v); (4) TNF-stim CM added at 10% (v/v); and (5) TNF-stim CM pre-incubated with a neutralizing anti-OPG antibody (AF459, R&D Systems) prior to addition to osteoclast cultures.

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

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-stimulated osteocyte-conditioned medium (CM) suppresses osteoclast differentiation partially via OPG. (A) Representative TRAP staining of osteoclasts generated under 5 conditions: (1) RANKL alone without any TNF-α source or osteocyte-CM (MLO-Y4−, TNF-α−); (2) direct TNF-α treatment in the absence of MLO-Y4 conditioned medium (MLO-Y4−, TNF-α+); (3) conditioned medium (CM) from unstimulated MLO-Y4 cells (MLO-Y4+, TNF-α−); (4) CM from TNF-α-stimulated MLO-Y4 cells collected after PBS wash and medium replacement (MLO-Y4+, TNF-α+); and (5) TNF-α-stimulated MLO-Y4 CM pre-incubated with a neutralizing anti-OPG antibody (AF459). Scale bars are 500 μm for low magnification and 200 μm for high magnification. (B) Dot plots of the percentage of spreading TRAP-positive osteoclasts for the conditions shown in (A) ( n = 3). (C) Quantitative RT-PCR analysis of Acp5 mRNA expression in osteoclast cultures under the conditions shown in (A). Acp5 expression levels were normalized to the reference gene ( n = 3). Data in (B and C) are individual values with mean ± SD; ** p < .01, **** p < .0001; ns indicates not significant by the one-way ANOVA with Tukey’s post-hoc test.

Article Snippet: For osteoclast differentiation assays, the following 5 conditions were compared (all containing 100 ng/mL RANKL): (1) no MLO-Y4 CM and no TNF-α (RANKL only; no TNF source); (2) TNF-α added directly to osteoclast cultures (TNF-α, no MLO-Y4); (3) Unstim CM added at 10% (v/v); (4) TNF-stim CM added at 10% (v/v); and (5) TNF-stim CM pre-incubated with a neutralizing anti-OPG antibody (AF459, R&D Systems) prior to addition to osteoclast cultures.

Techniques: Staining, Generated, Incubation, Quantitative RT-PCR, Expressing

Journal: BMC Oral Health

Article Title: Chronic intermittent hypobaric hypoxia attenuates root resorption following delayed tooth replantation by upregulating OPG/RANKL signaling pathway

doi: 10.1186/s12903-026-08135-7

Figure Lengend Snippet: CIHH downregulates RANKL expression and increases the OPG/RANKL ratio in delayed replanted teeth. A Immunohistochemistry staining showing OPG expression in the root and periapical tissue of NN and CIHH groups of delayed replanted teeth. Scale bar: 100 μm. B Immunohistochemistry staining showing RANKL expression in the root and periapical tissue of the NN and CIHH groups. Scale bar: 100 μm. C - D Representative Western blot bands ( C ) and quantitative analysis ( D ) showing OPG and RANKL protein expressions in the root and periapical tissue of NN and CIHH groups of delayed replanted teeth. D dentin; P: periodontal ligament; B: bone

Article Snippet: Membranes were blocked with 5% skim milk and incubated with a mouse anti-OPG primary antibody (1:1000, Huabio, China) or a rabbit anti-RANKL primary antibody (1:1000, Huabio, China) overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry, Staining, Western Blot

Journal: BMC Oral Health

Article Title: Chronic intermittent hypobaric hypoxia attenuates root resorption following delayed tooth replantation by upregulating OPG/RANKL signaling pathway

doi: 10.1186/s12903-026-08135-7

Figure Lengend Snippet: CIHH downregulates RANKL expression and increases the OPG/RANKL ratio in delayed replanted teeth. A Immunohistochemistry staining showing OPG expression in the root and periapical tissue of NN and CIHH groups of delayed replanted teeth. Scale bar: 100 μm. B Immunohistochemistry staining showing RANKL expression in the root and periapical tissue of the NN and CIHH groups. Scale bar: 100 μm. C - D Representative Western blot bands ( C ) and quantitative analysis ( D ) showing OPG and RANKL protein expressions in the root and periapical tissue of NN and CIHH groups of delayed replanted teeth. D dentin; P: periodontal ligament; B: bone

Article Snippet: The sections were then incubated overnight at 4 °C with primary antibodies: a mouse anti-OPG antibody (1:1000, Huabio, China) or a rabbit anti-RANKL antibody (1:1000, Huabio, China).

Techniques: Expressing, Immunohistochemistry, Staining, Western Blot