Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A&B) The effect of IL-1β on the mRNA expression of chemokine receptors in Tca8113 (A) and Hep2 (B) cells. Cells were treated with 20 ng/ml IL-1β for 24 h. The mRNA levels of CXCR4, CCR6 and CCR7 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (C) Quantitative CXCR4 mRNA expression in (A). * P < 0.05 compared with the non-treated group. (D) Time course of CXCR4 mRNA expression in response to IL-1β stimulation. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. The mRNA levels of CXCR4 were detected by RT-PCR. β-actin mRNA levels were measured as loading controls. (E) The quantitative data corresponding to (D). * P < 0.05 compared with the non-treated group. (F) The effect of IL-1β on CXCR4 protein expression. Tca8113 cells were treated with the indicated concentrations of IL-1β for 24 h. CXCR4 protein expression was detected by FACS. (G) The effect of IL-1β on SDF-1α-induced cell migration. Tca8113 cells were treated with the indicated concentrations of IL-1β for 24 h. Cell migration in response to medium or 20 ng/ml SDF-1α was measured by the Transwell assay. * P < 0.05 compared with control groups. (I) The transwell assay showed cell migration in response to 20 ng/ml SDF-1α after treatment with the indicated concentrations of IL-1β for 24 h (Scale bars: 200 μM).

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Migration, Transwell Assay, Control

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A) The expression of IL-1 receptors in Tca8113 cells. Cells were treated with the indicated concentrations of IL-1β for 24 h. The mRNA levels of IL-1R1 and IL-1RII were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (B) The expression of IL-1 receptors in Hep2. Cells were treated as described in (A). The mRNA levels of IL-1R1 and IL-1R2 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (C) The effect of IL-1β on IL-1R1 expression. Tca8113 cells were treated with the indicated concentrations of IL-1β for 24 h. The protein levels of IL-1R1 were measured by western blot. β-actin protein levels were measured as loading controls. (D&E) The effect of IL-1Ra on IL-1β-induced CXCR4 mRNA up-regulation. Tca8113 cells, pre-treated with the indicated concentrations of IL-1Ra for 1 h, were treated with or without 20 ng/ml IL-1β for 1 h. The mRNA levels of CXCR4 were measured by RT-PCR (D) and qRT-PCR (E). * P < 0.05 compared with the IL-1β-treated group. (F) The effect of IL-1Ra on IL-1β-induced CXCR4 protein up-regulation. Tca8113 cells, pre-treated with the indicated concentrations of IL-1Ra for 1 h, were treated with or without 20 ng/ml IL-1β for 24 h. The protein expression of CXCR4 was measured by FACS. (G) The effect of RNA interference on the expression of IL-1R1 protein. Tca8113 cells, transfected with non-specific shRNA (Nssi) or with IL-1R1 shRNA (IL-1R1si), were treated with the indicated concentrations of IL-1β for 24 h. The expression of IL-1R1 protein was measured by western blot. β-actin protein levels were measured as loading controls. (H) The effect of IL-1R1 down-regulation on CXCR4 mRNA expression. Non-specific shRNA (Nssi) or IL-1R1 shRNA (IL-1R1si) transfected Tca8113 cells were treated with medium or 20 ng/ml IL-1β for 24. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (I) The quantitative data corresponding to (H). * P < 0.05 compared with the Nssi group.

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Transfection, shRNA

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A) The effect of IL-1β on mRNA levels of IL-1β and TNF-α. Tca8113 cells were treated with the indicated concentrations of IL-1β for 24 h. The mRNA levels of IL-1β and TNF-α were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (B) IL-1β quantitative mRNA levels in (A). * P < 0.05 compared with the non-treated control. (C) Time-course of IL-1β mRNA expression in response to IL-1β treatment. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. The mRNA levels of IL-1β were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (D) IL-1β quantitative mRNA levels in (C). * P < 0.05 compared with the non-treated group. (E) The effect of IL-1Ra on IL-1β-induced IL-1β mRNA expression. Tca8113 cells, pre-treated with the indicated concentrations of IL-1Ra for 1 h, were stimulated with 20 ng/ml IL-1β for 24 h. The mRNA levels of IL-1β were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (F) IL-1β quantitative mRNA levels in (E). * P < 0.05 compared with the non-treated group. (G) The sustained effect of IL-1β on the expression of IL-1β and CXCR4. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated number of days. The mRNA levels of IL-1β and CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (H-I) Quantitative data of IL-1β (H) and CXCR4 (I) in (G). * P < 0.05 compared with the non-treated groups.

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Expressing

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A) Time-dependent activation of Notch by IL-1β. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. Activated Notch NCID fragments were detected by western blot. β-actin protein levels were measured as loading controls. (B) Dose dependent activation of Notch by IL-1β treatment for 1 h. (C) The effect of IL-1β on Hes1 mRNA levels. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. The mRNA levels of the Notch1 targeting gene Hes1 were measured by qRT-PCR. * P < 0.05 compared with the control group. (D) The effect of Notch inhibition on CXCR4 expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of Notch inhibitor L685458 for 30 min, were treated with 20 ng/ml IL-1β for 1 h. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (E) Quantitative data of CXCR4 expression in (D). * P < 0.05 compared with IL-1β-treated alone group. (F) The effect of Notch inhibition on IL-1β expression induced by IL-1β. Cells were treated as described in (D). The mRNA levels of IL-1β were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (G) Quantitative data of IL-1β expression in (F). * P < 0.05 compared with the IL-1β-treated alone group. (H) The effect of Notch inhibition on long-term CXCR4 mRNA expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of Notch inhibitor L685458 for 30 min, were treated with 20 ng/ml IL-1β for 24 h. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (I) The effect of Notch inhibition on CXCR4 protein expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of the Notch inhibitor L685458 for 30 min, were treated with 20 ng/ml IL-1β for 24 h. The protein levels of CXCR4 were measured by western blot. β-actin protein levels were measured as loading controls.

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Control, Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A) The effect of IL-1β on the activation of MAPKs. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. The phosphorylation levels of MAPKs were measured by western blot. β-actin protein levels were measured as loading controls. (B) The effect of IL-1β on the activation of IκB-α. Tca8113 cells were treated as described in (A). IκB-α protein levels were measured by western blot. β-actin protein levels were measured as loading controls. (C) The effect of ERK inhibition on IL-1β-induced CXCR4 expression. Tca8113 cells, pre-treated with the indicated concentrations of U0126 for 30 min, were stimulated with 20 ng/ml IL-1β for 1 h. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (D) Quantitative data of (C). * P < 0.05 compared with the IL-1β-treated group. (E) The effect of ERK inhibition on IL-1β-induced IL-1β mRNA expression. Tca8113 cells were treated as described in (C). The mRNA levels of IL-1β were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (F) Quantitative data of (E). * P < 0.05 compared with the IL-1β-treated group. (G) The effect of ERK inhibition on long-term CXCR4 mRNA expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of ERK inhibitor U0126 for 30 min, were treated with 20 ng/ml IL-1β for 24 h. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (I) The effect of ERK inhibition on CXCR4 protein expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of ERK inhibitor U0126 for 30 min, were treated with 20 ng/ml IL-1β for 24 h. The protein levels of CXCR4 were measured by western blot. β-actin protein levels were measured as loading controls.

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular Pain

Article Title: CXCR4 signaling in macrophages contributes to periodontal mechanical hypersensitivity in Porphyromonas gingivalis -induced periodontitis in mice

doi: 10.1177/1744806916689269

Figure Lengend Snippet: Involvement of CXCR4 signaling in infiltration of macrophages in gingival tissue following P.g.-L treatment. (a) CXCR4-IR and F4/80-IR cells in gingival tissue on day 2 following sham or P.g.-L treatment with vehicle or anti-CXCR4 neutralizing antibody. The arrows indicate CXCR4-IR and F4/80-IR cells in gingival tissue. The mean number of CXCR4-IR macrophages (b) and macrophages (c) in gingival tissue on day 2 after sham or P.g.-L treatment with vehicle or anti-CXCR4 neutralizing antibody. Data represent mean ± SEM. * p < 0.05 (n = 5 in each; one-way ANOVA followed by Tukey’s multiple-comparison test).

Article Snippet: Under light anesthesia with 2% isoflurane, anti-CXCR4 neutralizing antibody (1 μL, 50 μg/mL; cat. MAB21651; R&D Systems) dissolved in 0.01 M PBS was administered subcutaneously into the gingival tissue for 12 successive days (day 0 to day 11) in mice subjected to P.g.-L treatment.

Techniques: Comparison

Journal: Molecular Pain

Article Title: CXCR4 signaling in macrophages contributes to periodontal mechanical hypersensitivity in Porphyromonas gingivalis -induced periodontitis in mice

doi: 10.1177/1744806916689269

Figure Lengend Snippet: Involvement of CXCR4 signaling in mechanical sensitivity and NO production in gingival tissue following P.g.-L treatment. (a) Changes in mechanical sensitivity measured in the gingival tissue following sham or P.g.-L treatment with vehicle or anti-CXCR4 neutralizing antibody. Data represent mean ± SEM. Pre: three days before inoculation. ** p < 0.01 versus sham with vehicle (two-way ANOVA followed by Bonferroni’s multiple-comparison test). (b) Quantitative analysis of nitrate and nitrite on day 2 after sham or P.g.-L treatment with vehicle or anti-CXCR4 neutralizing antibody. Data represent mean ± SEM. * p < 0.05 (n = 6 or 7 in each; one-way ANOVA followed by Newman-Keuls’s multiple-comparison test).

Article Snippet: Under light anesthesia with 2% isoflurane, anti-CXCR4 neutralizing antibody (1 μL, 50 μg/mL; cat. MAB21651; R&D Systems) dissolved in 0.01 M PBS was administered subcutaneously into the gingival tissue for 12 successive days (day 0 to day 11) in mice subjected to P.g.-L treatment.

Techniques: Comparison

Journal: Molecular Pain

Article Title: CXCR4 signaling in macrophages contributes to periodontal mechanical hypersensitivity in Porphyromonas gingivalis -induced periodontitis in mice

doi: 10.1177/1744806916689269

Figure Lengend Snippet: Effect of NO on mechanical sensitivity in the gingival tissue following P.g.-L treatment. (a) Time course of change in mechanical sensitivity of gingival tissue after administration of L-arginine in naive mice. Data represent mean ± SEM. Pre: 3 hours before administration. ** p < 0.01 versus vehicle (n = 6 in each; two-way repeated-measures ANOVA followed by Bonferroni’s multiple-comparison test). (b) Time course of change in mechanical sensitivity in gingival tissue after administration of vehicle or L-NAME in mice treated with P.g.-L with vehicle or anti-CXCR4 neutralizing antibody. Data represent mean ± SEM. Pre: three days before inoculation. ** p < 0.01 versus P.g.-L with vehicle + vehicle (n = 7 in each; two-way repeated-measures ANOVA followed by Bonferroni’s multiple-comparison test).

Article Snippet: Under light anesthesia with 2% isoflurane, anti-CXCR4 neutralizing antibody (1 μL, 50 μg/mL; cat. MAB21651; R&D Systems) dissolved in 0.01 M PBS was administered subcutaneously into the gingival tissue for 12 successive days (day 0 to day 11) in mice subjected to P.g.-L treatment.

Techniques: Comparison