Journal: Drug Design, Development and Therapy

Article Title: Sinomenine Inhibits Orthodontic Tooth Movement and Root Resorption in Rats and Enhances Osteogenic Differentiation of PDLSCs

doi: 10.2147/DDDT.S379468

Figure Lengend Snippet: Results of immunohistochemical staining. ( A – B ) The result of TNF-α ( A ) and RANKL ( B ) immunostaining of the maxillary first molar indicated that the expression of TNF-α and RANKL decreased in sinomenine-treated groups. ( C – E ) The results of OPG ( C ), RUNX2 ( D ) and OCN ( E ) IHC staining indicated that the expression of OPG, RUNX2 and OCN increased. Scale Bar: 20 μm. Data are expressed as mean ± SD (* P<0.05, **P<0.01, ***P<0.001).

Article Snippet: The blots were probed with primary antibodies overnight at 4 °C to ALP (Huabio, Hangzhou, China), RUNX2 (Proteintech, Hubei, China), OPG (Abcam, Cambridge, UK), RANKL and GAPDH (Proteintech, Hubei, China).

Techniques: Immunohistochemical staining, Staining, Immunostaining, Expressing, Immunohistochemistry

Journal: Drug Design, Development and Therapy

Article Title: Sinomenine Inhibits Orthodontic Tooth Movement and Root Resorption in Rats and Enhances Osteogenic Differentiation of PDLSCs

doi: 10.2147/DDDT.S379468

Figure Lengend Snippet: Effect of sinomenine on proliferation and osteogenesis of PDLSCs. ( A ) PDLSCs were treated with 0, 0.1 M, 0.2 M, 0.5 M, 1.0 M, 2.0 M sinomenine for 0, 1, 3, and 5 days, and the growth curves were depicted according to the results of CCK-8 assay. ( B ) 0.1 M sinomenine stimulated the proliferation of PDLSCs, while 1.0 M and 2.0 M sinomenine markedly inhibited the proliferation of PDLSCs on day 5. ( C ) Mineral deposition was observed by Alizarin red staining. Scale bar: 200 μm. ( D ) The relative amount of mineralized nodule was assessed based on the absorbance at 562 nm. ( E ) ALP activity was enhanced by 0.1 M and 0.5 M sinomenine at day 7. ( F and G ) The mRNA and protein expression levels of ALP ( F ) and RUNX2 ( G ) in sinomenine-treated groups for 7 days. Data are presented as mean ± SD (**P<0.01, ***P<0.001, ****P<0.0001).

Article Snippet: The blots were probed with primary antibodies overnight at 4 °C to ALP (Huabio, Hangzhou, China), RUNX2 (Proteintech, Hubei, China), OPG (Abcam, Cambridge, UK), RANKL and GAPDH (Proteintech, Hubei, China).

Techniques: CCK-8 Assay, Staining, Activity Assay, Expressing

Journal: Oncotarget

Article Title: Loss of RUNX3 expression inhibits bone invasion of oral squamous cell carcinoma

doi: 10.18632/oncotarget.14071

Figure Lengend Snippet: ( A ) shCTRL or shRUNX3 Ca9.22 cells (1 × 10 5 cells/dish) were treated with 10 ng/ml TGF-β for 24 h. The expression level of PTHrP was examined in the total lysates with a Western blot analysis. GAPDH served as a loading control. ( B ) shCTRL or shRUNX3 Ca9.22 cells (1 × 10 3 cells/well) were treated with 10 ng/ml TGF-β for 24 h. PTHrP levels in the conditioned media were analyzed with a ELISA kit. The results are expressed as the mean ± SE. * P < 0.05 versus shCTRL cells without TGF-β. ( C ) hFOB1.19 osteoblastic cells (1 × 10 6 cells/dish) were incubated with the respective shCTRL or shRUNX3 Ca9.22 cell-derived conditioned media for 6 h. The expression levels of RANKL and OPG in osteoblastic cells were determined with a Western blot analysis. GAPDH served as a loading control. ( D ) hFOB1.19 cells (1 × 10 4 cells/well) were incubated for 24 h with or without conditioned media derived from shCTRL or shRUNX3 Ca9.22 cells. The culture media from hFOB1.19 cells were analyzed for the secreted levels of RANKL and OPG with ELISA kits. The results are expressed as the mean ± SE. * P < 0.05 versus control (without conditioned media), # P < 0.05 versus conditioned media derived from untreated shCTRL cells.

Article Snippet: The culture media from hFOB1.19 cells were analyzed for the secreted levels of RANKL and OPG using RANKL ELISA kit (EIAab, Wuhan, China) and OPG ELISA kit (Boster Biological Technology, CA, USA) according to the manufacturer's instructions.

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

Journal: Oncotarget

Article Title: MicroRNA-106b inhibits osteoclastogenesis and osteolysis by targeting RANKL in giant cell tumor of bone

doi:

Figure Lengend Snippet: A. Prediction of the miR-106b target site in RANKL 3′UTR in humans and animals. Schematic diagram of miR-106b with WT and MUT 3′UTR regions of RANKL are shown in complementary pairing. Mutated nucleotides are labeled. B. Determination of the effect of agomir-Control, agomiR-106b on luciferase activity in HEK293 cells transfected with either WT-RANKL 3′UTR reporter or mutant RANKL 3′ UTR reporter. Firefly luciferase values are normalized for Renilla luciferase. C. qRT-PCR analysis of RANKL mRNA levels in GCTSCs and MG63 cells treated with agomiR-106b or antagomiR-106b and their corresponding negative controls. D. , E. ELISA and Western blot analysis of RANKL protein level. F. - H. qRT-PCR analysis of the mRNA levels of MMP2, IL-8 and TWIST. * P < 0.05.

Article Snippet: ELISA assay was performed with Uscn (Uscn Life Science Inc., Wuhan, China) ELISA kit for human RANKL (SCA855Hu) following the manufacturer's instructions.

Techniques: Labeling, Control, Luciferase, Activity Assay, Transfection, Mutagenesis, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Oncotarget

Article Title: MicroRNA-106b inhibits osteoclastogenesis and osteolysis by targeting RANKL in giant cell tumor of bone

doi:

Figure Lengend Snippet: A. qRT-PCR analysis of miR-106b level 48 h after transfection. B. qRT-PCR analysis of mRNA level of RANKL. C. , D. Western blot and ELISA analysis of RANKL protein level. E. - G. qRT-PCR analysis of mRNA levels of IL-8, MMP2 and TWIST. (H-M) Mouse BMMs were seeded on dentin slices or empty wells and cultured with conditional medium containing MCFS (10 ng/mL) from OE-miR106b or OE-miR106b-Control GCTSCs for 7 days. H. The number of TRAP positive osteoclasts per well. I. TRAP staining of BMM cells. J. Phalloidin-Rhodamine staining of BMM cells. K. TRAP activity assay of BMM cells. L. Dentine slices stained with Mayer's hematoxylin after removal of cells. M. The number of pits per dentine slice. * P < 0.05.

Article Snippet: ELISA assay was performed with Uscn (Uscn Life Science Inc., Wuhan, China) ELISA kit for human RANKL (SCA855Hu) following the manufacturer's instructions.

Techniques: Quantitative RT-PCR, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Staining, Activity Assay

Journal: ACS Omega

Article Title: Rapamycin Inhibits Osteoclastogenesis and Prevents LPS-Induced Alveolar Bone Loss by Oxidative Stress Suppression

doi: 10.1021/acsomega.3c01289

Figure Lengend Snippet: Rapamycin inhibited OC differentiation in a dose-dependent manner. (A) CCK-8 assay. (B) TRAP staining of the RAW 264.7 cells and BMMs. Scale bar: 200 μm. (C,D) Quantitation of RANKL-induced TRAP-positive cells and multinucleated cells with/without denosumab or rapamycin. n = 6; versus 5 days RANKL-induced group; **** P < 0.0001. (E) mRNA levels of mature OC function-related markers in BMMs treated with/without denosumab or rapamycin, for 5 days, respectively. n = 3; versus 5 days RANKL-induced group; **** P < 0.0001. Abbreviations: CTSK: Cathepsin K; ACP5: tartrate resistant acid phosphatase 5; MMP-9: matrix metalloproteinase-9; Nfatc1: nuclear factor of activated T cells 1; 5d: 5 days RANKL induction group; Deno: denosumab treatment group.

Article Snippet: The Elisa kits for RANKL, OPG (osteoprotegerin), TRACP 5b, TNF-α, IL-1β, and IL-6, as well as the biochemical kits for alkaline phosphatase (ALP), malondialdehyde (MDA), superoxide dismutase (SOD), and nitric oxide (NO), were purchased from Nanjing Jiancheng (Nanjing, China).

Techniques: CCK-8 Assay, Staining, Quantitation Assay

Journal: ACS Omega

Article Title: Rapamycin Inhibits Osteoclastogenesis and Prevents LPS-Induced Alveolar Bone Loss by Oxidative Stress Suppression

doi: 10.1021/acsomega.3c01289

Figure Lengend Snippet: Rapamycin significantly suppressed the level of oxidative stress during OC formation and upregulated the Nrf2/GCLC s ignaling pathway in RAW 264.7 cells in a dose-dependent manner. (A) ROS levels in RAW 264.7 cells and BMMs using DCFH2-DA. Scale bar: 100 μm. (B) Mitochondrial superoxide levels in RAW 264.7 cells and BMMs using MitoSOX. Scale bar: 100 μm. (C) Quantitative analysis of DCFH2-DA fluorescence intensity. (D) Quantitative analysis of MitoSOX fluorescence intensity. (E) Autophagy level in RAW 264.7 cells and BMMs using MDC. Scale bar: 100 μm. (F) Quantitative analysis of MDC fluorescence intensity. (G) Expression levels of p -mTOR, mTOR, Nrf2, GCLC, HO-1, LC3B, and p62, relative to β-actin, in RAW 264.7 cells in the presence of 0/50/100/200 nM rapamycin, after 5 days of OC differentiation. (H) Protein intensity was analyzed by Image J software. (C, D, F, H) n = 3; versus the 5 day RANKL-induced group; ns, no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The Elisa kits for RANKL, OPG (osteoprotegerin), TRACP 5b, TNF-α, IL-1β, and IL-6, as well as the biochemical kits for alkaline phosphatase (ALP), malondialdehyde (MDA), superoxide dismutase (SOD), and nitric oxide (NO), were purchased from Nanjing Jiancheng (Nanjing, China).

Techniques: Fluorescence, Expressing, Software

Journal: ACS Omega

Article Title: Rapamycin Inhibits Osteoclastogenesis and Prevents LPS-Induced Alveolar Bone Loss by Oxidative Stress Suppression

doi: 10.1021/acsomega.3c01289

Figure Lengend Snippet: Rapamycin inhibiteds OC formation and function by oxidative suppression. (A,B) The cytotoxicity of TBHP in the presence of rapamycin with/without RANKL stimulation. n = 3; versus 100/200 nM rapamycin; ns, no significance, * P < 0.05, ** P < 0.01. (C) ROS levels and superoxide levels in RAW 264.7 cells in the presence of TBHP and rapamycin during RANKL stimulation. Scale bar: 100 μm. (D,E) Quantitative analysis of DCFH2-DA and MitoSOX fluorescence intensity. n = 3; versus 5 days of induction with 200 nM rapamycin; ns, no significance, ** P < 0.01. Scale bar: 100 μm. (F) TRAP staining of the RAW 264.7 cells. Scale bar: 100 μm. (G) Quantitative analysis of the number of multinuclear cells of mature OC from RAW 264.7 cells. n = 6; versus 5 days of induction with 200 nM rapamycin; * P < 0.05, ** P < 0.01, **** P < 0.0001. (H) OC mature and functional genes of RAW 264.7 cells treated with or without 20/50 μM TBHP in the presence or absence of 200 nM rapamycin for 5 days. n = 3; versus 5 days of induction with 200 nM rapamycin; * P < 0.05, ** P < 0.01, **** P < 0.0001. (I) Representative images of bone resorption measurement. Scale bar: 100 μm. (J) Quantitative analysis of bone resorption area. n = 3; versus 7 days induction group; * P < 0.05, **** P < 0.0001.

Article Snippet: The Elisa kits for RANKL, OPG (osteoprotegerin), TRACP 5b, TNF-α, IL-1β, and IL-6, as well as the biochemical kits for alkaline phosphatase (ALP), malondialdehyde (MDA), superoxide dismutase (SOD), and nitric oxide (NO), were purchased from Nanjing Jiancheng (Nanjing, China).

Techniques: Fluorescence, Staining, Functional Assay

Journal: ACS Omega

Article Title: Rapamycin Inhibits Osteoclastogenesis and Prevents LPS-Induced Alveolar Bone Loss by Oxidative Stress Suppression

doi: 10.1021/acsomega.3c01289

Figure Lengend Snippet: Rapamycin alleviates the bone resorption serum indicator associated with periodontitis in vivo and decreases the level of oxidative stress and inflammatory factor secretion. (A) Serum levels of RANKL, OPG, TRACP5b, and ALP. (B) Total MDA, SOD, and NO of rat serum. (C) Serum levels of IL-1β, IL-6, and TNF-α. (A, B, C) n = 5, versus model group; * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001.

Article Snippet: The Elisa kits for RANKL, OPG (osteoprotegerin), TRACP 5b, TNF-α, IL-1β, and IL-6, as well as the biochemical kits for alkaline phosphatase (ALP), malondialdehyde (MDA), superoxide dismutase (SOD), and nitric oxide (NO), were purchased from Nanjing Jiancheng (Nanjing, China).

Techniques: In Vivo