Journal: International Journal of Molecular Sciences

Article Title: Interleukin-23 Mediates Osteoclastogenesis in Collagen-Induced Arthritis by Modulating MicroRNA-223

doi: 10.3390/ijms23179718

Figure Lengend Snippet: Endogenous expression of IL-23p19 and clinical assessments in the LVshIL-23p19-treated mice with collagen-induced arthritis (CIA). ( A ) Reverse transcription polymerase chain reaction (RT-PCR) analysis of IL-23p19 expression in the synovial tissue of mice with CIA (Day 10, 21, 42). Each lane represents pooled samples from 3 animals. Results are representative of two independent experiments. ( B ) Amelioration in mice with CIA by evaluating with arthritis score after the LVshIL-23p19 treatment. Mice that had been immunized with collagen on days 0 and 21 were injected intraperitoneally with LVshIL-23p19, LVshLuc and medium on day 21, respectively. The arrow indicates the time at which the lentiviral vectors were injected. Each value shown represents the mean ± SEM ( n = 6). ( C ) Representative images of the ankle sections by hematoxylin and eosin staining on day 45. ( D ) Representative images and quantification of the levels of phosphorylated signal transducers and activators of transcription 4 (p-STAT4) by immunohistochemical staining on day 45. Each value shown represents the mean ± SEM ( n = 3). ( E ) Representative images of the levels of miR-223 by in situ hybridization on day 45. Scale bars represent 500, 200 and 50 μm in ×40, ×100 and ×400 magnifications, respectively. Each value shown represents the mean ± SEM ( n = 3). Black boxed areas were shown at higher magnification in the panels (×400) beneath them. Results are representative of two independent experiments.

Article Snippet: Briefly, IL-23-treated RAW264.7 cells were formaldehyde crossed-linked, and cell lysates were sonicated to shear the DNA to an average length of 500 bp followed by immunoprecipitation with rabbit anti-phosphorylated STAT4 (ChIP grade, Cell Signaling Technology, Danvers, MA, USA) antibody and normal rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA) in combination with protein G agarose beads.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Injection, Staining, Immunohistochemical staining, In Situ Hybridization

Journal: International Journal of Molecular Sciences

Article Title: Interleukin-23 Mediates Osteoclastogenesis in Collagen-Induced Arthritis by Modulating MicroRNA-223

doi: 10.3390/ijms23179718

Figure Lengend Snippet: Association of IL-23 signaling with miR-223 in osteoclastogenesis. ( A ) Tartrate-resistant acid phosphatase (TRAP) staining and quantification in IL-23 (5 and 20 ng/mL) treated bone marrow-derived macrophages (BMMs). Each value shown represents the mean ± SEM ( n = 4). ( B ) TRAP staining and quantification in LVmiR-223T and LVshLuc-infected BMMs (MOI = 0.05 and 0.5) in response to IL-23 stimulation (20 ng/mL). Each value shown represents the mean ± SEM ( n = 3). ( C ) TRAP staining and quantification in LVshSTAT4#1 (TRCN0000081638), LVshSTAT4#2 (TRCN0000081639) and LVshLuc-infected BMMs (MOI = 1 and 5) in response to IL-23 stimulation (20 ng/mL). Scale bars represent 200 μm in ×100 magnifications. Each value shown represents the mean ± SEM ( n = 3). ( D ) Binding of phosphorylated STAT4 to the miR-223 promoter in IL-23-treated RAW264.7 cells by determining with quantitative chromatin immunoprecipitation (qChIP) assay. Each value shown represents the mean ± SEM ( n = 3). ( E ) Expression of miR-223 in BMMs treated with various concentrations of IL-23 for 12 h, as determined by quantitative RT-PCR (qRT-PCR). Each value shown represents the mean ± SEM ( n = 3). ( F ) Expression of miR-223 in LVshLuc, LVshSTAT4#1 (TRCN0000081638) and LVshSTAT4#2 (TRCN0000081639)-transduced BMMs upon stimulating with IL-23 (20 ng/mL) for 12 h, as determined by qRT-PCR. Each value shown represents the mean ± SEM ( n = 3). Red boxed areas are shown at higher magnification in the panels beneath them. Results are representative of two independent experiments.

Article Snippet: Briefly, IL-23-treated RAW264.7 cells were formaldehyde crossed-linked, and cell lysates were sonicated to shear the DNA to an average length of 500 bp followed by immunoprecipitation with rabbit anti-phosphorylated STAT4 (ChIP grade, Cell Signaling Technology, Danvers, MA, USA) antibody and normal rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA) in combination with protein G agarose beads.

Techniques: Staining, Derivative Assay, Infection, Binding Assay, Chromatin Immunoprecipitation, Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Interleukin-23 Mediates Osteoclastogenesis in Collagen-Induced Arthritis by Modulating MicroRNA-223

doi: 10.3390/ijms23179718

Figure Lengend Snippet: Lisofylline (LSF) treatment in BMMs and mice with CIA. ( A ) TRAP staining and quantification in LSF (50 and 100 μM)-treated BMMs upon stimulating with IL-23 (20 ng/mL). Each value shown represents the mean ± SEM ( n = 3). Red boxed areas are shown at higher magnification in the panels next to them. Scale bars represent 200 μm in ×100 magnifications. ( B ) Expression of miR-223 in BMMs treated with various concentrations of LSF in response to IL-23 (20 ng/mL) stimulation for 12 h, as determined by qRT-PCR. Each value shown represents the mean ± SEM ( n = 3). ( C ) Arthritis score and incidence in mice with CIA through intraperitoneal injections of LSF (50 mg/kg) and PBS daily from day 22 to 36. Arrows indicate the time at which LSF was injected. Each value shown represents the mean ± SEM (PBS: n = 6, LSF: n = 6). ( D ) Representative images of the ankle sections by hematoxylin and eosin staining on day 40. Scale bars represent 200 and 50 μm in ×100 and ×400 magnifications, respectively. Each value shown represents the mean ± SEM ( n = 6). Black boxed areas were shown at higher magnification in the panels (×400) beneath them. Expression of ( E ) miR-223, ( F ) phospho-STAT4 (p-STAT4) and ( G ) the number of TRAP-positive cells in joint extracts of LSF and PBS-injected mice with CIA, as determined by qRT-PCR. Each value shown represents the mean ± SEM ( n = 6). Results are representative of two independent experiments.

Article Snippet: Briefly, IL-23-treated RAW264.7 cells were formaldehyde crossed-linked, and cell lysates were sonicated to shear the DNA to an average length of 500 bp followed by immunoprecipitation with rabbit anti-phosphorylated STAT4 (ChIP grade, Cell Signaling Technology, Danvers, MA, USA) antibody and normal rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA) in combination with protein G agarose beads.

Techniques: Staining, Expressing, Quantitative RT-PCR, Injection

Journal: International Journal of Molecular Sciences

Article Title: Interleukin-23 Mediates Osteoclastogenesis in Collagen-Induced Arthritis by Modulating MicroRNA-223

doi: 10.3390/ijms23179718

Figure Lengend Snippet: A schematic overview of silencing IL-23-STAT4-miR-223 signaling axis in preventing osteoclastogenesis. IL-23p19 can bind to the IL-23 receptor (IL-23R), which causes phosphorylation and translocation of STAT4 into the nucleus. P-STAT4 further conjugates with the promoter region of primary miR-223 (pri-miR-223) and the transactivated pri-miR-223 translocates into the cytosol to become mature miR-223 (miR-223) and mediates osteoclastogenesis. The signaling axis, IL-23-STAT4-miR-223 is proved to mediate osteoclastogenesis by introducing IL-23-stimulated BMMs and mouse CIA model with LVshIL-23p19, LVshSTAT4, LSF, and LVmiR-223T that target IL-23p19, STAT4, and miR-223.

Article Snippet: Briefly, IL-23-treated RAW264.7 cells were formaldehyde crossed-linked, and cell lysates were sonicated to shear the DNA to an average length of 500 bp followed by immunoprecipitation with rabbit anti-phosphorylated STAT4 (ChIP grade, Cell Signaling Technology, Danvers, MA, USA) antibody and normal rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA) in combination with protein G agarose beads.

Techniques: Translocation Assay

Journal: PLoS ONE

Article Title: Mangiferin Attenuates Th1/Th2 Cytokine Imbalance in an Ovalbumin-Induced Asthmatic Mouse Model

doi: 10.1371/journal.pone.0100394

Figure Lengend Snippet: (A) The levels of p-STAT6 and p-STAT4 in lung tissues were measured by western blot. (B) Bar plot of the average relative density of p-STAT4 and p-STAT6. (C) The levels of GATA3 and T-bet in lung tissues were measured by western blot. (D) Bar plot of the average relative density of GATA3 and T-bet. (E) The expression of STAT6 in lung tissues was detected by immunohistochemistry (magnification 400×), only a representative picture is shown for each group. (a) Normal; (b) Model; (c) Mangiferin 50 mg/kg, (d) Mangiferin 100 mg/kg, (e) Mangiferin 200 mg/kg, (f) Dexamethasone 1.25 mg/kg. Three independent experiments were examined (12 mice in each group of one experiment), Data are expressed as means ± SDs. Compared with model group: * P <0.05; ** P <0.01.

Article Snippet: Anti-STAT4, STAT6, phosphorylated STAT4, phosphorylated STAT6, GAPDH, antibodies were purchased from Cell Signaling Technology (Danvers, USA).

Techniques: Western Blot, Expressing, Immunohistochemistry