Journal: International journal of molecular sciences

Article Title: Dual Role of Interleukin-20 in Different Stages of Osteoclast Differentiation and Its Osteoimmune Regulation during Alveolar Bone Remodeling.

doi: 10.3390/ijms24043810

Figure Lengend Snippet: Figure 1. IL-20 accelerated OVX-induced bone loss in orthodontic tooth movement by promoting osteoclastogenesis. (A) Scheme illustrating the establishment of orthodontic tooth movement. (B) The distance of orthodontic tooth movement in the Control group, Force group, and OVX + Force group on day 16. (C) TRAP and Immunohistochemical staining showed that TRAP and IL-20 -positive cells and in the Control group, Force group, and OVX + Force group. Green triangles showed TRAP- positive osteoclasts. Red triangles showed IL-20-positive cells. T: tooth, PL: periodontal ligament, AB: alveolar bone. (D) The quantification of TRAP-positive osteoclasts in the Control group, Force group, and OVX + Force group. (E) Immunohistochemical staining and semiquantification of IL-20 in the Control group, Force group, and OVX + Force group. (F) Double-labelled immunofluorescence staining showed that, in the context of orthodontic force, the expression levels of IL-20 and osteoclast marker protein TRAP increased in the first molar periodontal ligament. (G) The distance of orthodon- tic tooth movement in the OVX group, OVX + Force group, and OVX + Force + risedronate group on day 16. (H) TRAP and immunohistochemical staining showed that TRAP and IL-20 -positive cells and in the OVX group, OVX + Force group, and OVX + Force + risedronate group. (I) The quantification of TRAP-positive osteoclasts in the OVX group, ovariectomy + Force group, and Ovariectomy + Force + risedronate group. (J) Immunohistochemical staining and semiquantification of IL-20 in the OVX group, OVX + Force group, and OVX + Force + risedronate group. (K) Im- munofluorescence staining showed that the expression levels of IL-20 and osteoclast marker protein TRAP increased in the first molar periodontal ligament. * p < 0.05 vs. the control group. n = 6.

Article Snippet: After the treatment of IL-20, anti-IL-20 antibody, or TPCA-1 (MCE, Monmouth Junction, NJ, USA), we obtained protein from the samples with cold RIPA containing 1% protease inhibitor cocktail and 1% phosphatase inhibitors.

Techniques: Control, Immunohistochemical staining, Staining, Expressing, Marker

Journal: International journal of molecular sciences

Article Title: Dual Role of Interleukin-20 in Different Stages of Osteoclast Differentiation and Its Osteoimmune Regulation during Alveolar Bone Remodeling.

doi: 10.3390/ijms24043810

Figure Lengend Snippet: Figure 2. IL-20 promoted preosteoclast viability via MAPK pathway at the early stage of osteoclast differentiation. Cell viability was examined in M-CSF-induced preosteoclasts by a CCK8 assay. (A,B) The expression of IL-20 in M-CSF-induced preosteoclasts was determined by cellular im- munofluorescence staining on day 2. (C–F) The mRNA expression levels of IL-20, IL-20RA, IL-20RB, and IL-22RA1 in preosteoclasts were evaluated by qRT-PCR after 2 days of M-CSF treatment. (G) Cell viability detection in preosteoclasts treated with a gradient of concentrations of IL-20 on days 1, 3, 5, and 7. (H) Preosteoclasts were treated with 2 ng/mL IL-20 or anti-IL-20 antibody. The levels of phosphorylation of proteins in the IL-20-mediated signaling pathway, including p38, phos-pho-p38, ERK, phospho-ERK, JNK, and phospho-JNK, were analyzed using Western blotting. IL-20-block group meant that cells were treated with IL-20 and anti-IL-20 antibody. * p < 0.05 vs. the 0 ng/mL IL-20 group. ns p > 0.05 vs. the 0 ng/mL IL-20 group. n = 6.

Article Snippet: After the treatment of IL-20, anti-IL-20 antibody, or TPCA-1 (MCE, Monmouth Junction, NJ, USA), we obtained protein from the samples with cold RIPA containing 1% protease inhibitor cocktail and 1% phosphatase inhibitors.

Techniques: CCK-8 Assay, Expressing, Staining, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Blocking Assay

Journal: International journal of molecular sciences

Article Title: Dual Role of Interleukin-20 in Different Stages of Osteoclast Differentiation and Its Osteoimmune Regulation during Alveolar Bone Remodeling.

doi: 10.3390/ijms24043810

Figure Lengend Snippet: Figure 3. IL-20 had no effect on osteoclast formation at the early stage of osteoclast differentiation. (A) Scheme illustrating bone marrow-derived macrophages were cultured in osteoclast medium containing M-CSF and IL-20 or anti-IL-20 antibody at the early stage of osteoclast differentiation, and then induced with the presence of 10, 30, or 60 ng/mL RANKL. The control group included M-CSF-induced preosteoclasts induced with 10, 30, or 60 ng/mL RANKL. (B) TRAP staining was performed, and the number and size of TRAP-positive osteoclasts with more than three nuclei were quantified on day 6. IL-20-block group meant that cells were treated with IL-20 or anti-IL-20 antibody. (C) A bone resorption pit assay was performed to detect osteoclast function, and bone resorption pits were counted, and the area and number of bone resorption were quantified on day 6. IL-20- block group meant that cells were treated with IL-20 and anti-IL-20 antibody. (D) M-CSF-induced preosteoclasts were cultured in osteoclast medium containing different concentrations of IL-20 or anti-IL-20 antibody without RANKL. n = 6.

Article Snippet: After the treatment of IL-20, anti-IL-20 antibody, or TPCA-1 (MCE, Monmouth Junction, NJ, USA), we obtained protein from the samples with cold RIPA containing 1% protease inhibitor cocktail and 1% phosphatase inhibitors.

Techniques: Derivative Assay, Cell Culture, Control, Staining, Blocking Assay

Journal: International journal of molecular sciences

Article Title: Dual Role of Interleukin-20 in Different Stages of Osteoclast Differentiation and Its Osteoimmune Regulation during Alveolar Bone Remodeling.

doi: 10.3390/ijms24043810

Figure Lengend Snippet: Figure 4. IL-20 promoted RANKL-induced osteoclast differentiation and bone resorption function. (A) Scheme illustrating M-CSF-induced preosteoclasts were cultured in osteoclast medium containing IL-20 or anti-IL-20 antibody at the late stage of osteoclast differentiation in the presence of 10, 30, or 60 ng/mL RANKL. (B) TRAP staining was performed, and the number and size of TRAP-positive osteoclasts with more than three nuclei were quantified at the late stage of differentiation on day 6. The control group included M-CSF-induced preosteoclasts induced with 10, 30, or 60 ng/mL RANKL. IL-20-block group meant that cells were treated with IL-20 and anti-IL-20 antibody. (C) A resorption pit assay was performed to detect osteoclast function, and resorption pits were counted at the late stage of differentiation on day 6, and the area and number of bone resorption were quantified at the stage of differentiation. IL-20-block group meant that cells were treated with IL-20 and anti-IL-20 antibody. * p < 0.05 vs. the control group. n = 6.

Article Snippet: After the treatment of IL-20, anti-IL-20 antibody, or TPCA-1 (MCE, Monmouth Junction, NJ, USA), we obtained protein from the samples with cold RIPA containing 1% protease inhibitor cocktail and 1% phosphatase inhibitors.

Techniques: Cell Culture, Staining, Control, Blocking Assay

Journal: International journal of molecular sciences

Article Title: Dual Role of Interleukin-20 in Different Stages of Osteoclast Differentiation and Its Osteoimmune Regulation during Alveolar Bone Remodeling.

doi: 10.3390/ijms24043810

Figure Lengend Snippet: Figure 5. IL-20 promoted the expression of osteoclast-specific genes and proteins via NF-κB pathway during RANKL-induced osteoclast differentiation. Preosteoclasts were stimulated with IL-20 or NF-κB pathway inhibitor TPCA-1. (A,B) IL-20 promoted phospho-P65 nuclear translocation and was blocked by TPCA-1 in preosteoclasts after 1 h treatment, with or without RANKL. (C) The levels of phosphorylation for proteins in the NF-κB pathway, including the IKKα/β, IκB-α, and P65 proteins in preosteoclasts were detected using Western blotting. (D) The levels of activated proteins in preosteoclasts, including the RANK, TRAF6, c-Fos, and NFATc1 proteins without RANKL were detected using Western blotting. (E) The protein expression levels of TRAP, Cathepsin K, RANK, TRAF6, c-Fos and NFATc1 in osteoclasts were examined using Western blotting after 6 days of IL-20 treatment. (F–K) The mRNA expression levels of TRAP, Cathepsin K, MMP9, MT1-MMP, c-Fos, and NFATc1 in osteoclasts were detected by qRT-PCR after 6 days of IL-20 treatment. * p < 0.05 vs. the 0 ng/mL IL-20 group. n = 6.

Article Snippet: After the treatment of IL-20, anti-IL-20 antibody, or TPCA-1 (MCE, Monmouth Junction, NJ, USA), we obtained protein from the samples with cold RIPA containing 1% protease inhibitor cocktail and 1% phosphatase inhibitors.

Techniques: Expressing, Translocation Assay, Phospho-proteomics, Western Blot, Quantitative RT-PCR

Journal: International journal of molecular sciences

Article Title: Dual Role of Interleukin-20 in Different Stages of Osteoclast Differentiation and Its Osteoimmune Regulation during Alveolar Bone Remodeling.

doi: 10.3390/ijms24043810

Figure Lengend Snippet: Figure 6. Therapeutic effect of IL-20 on orthodontic tooth movement. (A,B) Micro-CT showed the distance of orthodontic tooth movement between first and second molars treated with local infusion of IL-20 or an-ti-IL-20 antibody, seven days after the application of orthodontic force. OTM + IL- 20-block group meant that rats were locally infused with anti-IL-20 antibody. (C,D) TRAP staining and the quantification of TRAP-positive osteoclasts in the OTM group, OTM + IL-20 group, and OTM + anti-IL-20 antibody group. Green triangles showed TRAP-positive osteoclasts. OTM + IL-20- block group meant that rats were locally infused with anti-IL-20 antibody. (E,F) Immunofluorescence staining showed that the expression levels of osteoclast marker protein RANK in the first molar periodontal ligament after the application of orthodontic force. OTM + IL-20-block group meant that rats were locally infused with anti-IL-20 antibody. (G,H) Immunofluorescence staining showed that the expression levels of MCP-1 and CD11b in the first molar periodontal ligament after the application of orthodontic force. * p < 0.05 vs. the control group. n = 6.

Article Snippet: After the treatment of IL-20, anti-IL-20 antibody, or TPCA-1 (MCE, Monmouth Junction, NJ, USA), we obtained protein from the samples with cold RIPA containing 1% protease inhibitor cocktail and 1% phosphatase inhibitors.

Techniques: Micro-CT, Blocking Assay, Staining, Expressing, Marker, Control