Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) Invasion of Ca9.22 and YD10B OSCC cells treated with CCL28 and/or TGF-β (mean ± SEM, n = 3). *P < 0.05 vs. cells without CCL28 and TGF-β; #P < 0.05, ##P < 0.005 vs. TGF-β–only–treated cells by 1-way ANOVA with multiple-comparisons test. (B) Invasion of Ca9.22 and YD10B OSCC cells with CCL28 and/or TGF-β into the CAMs of fertilized eggs (mean ± SEM, n = 3). Representative images of CAM. Scale bars: 100 μm. Cells invaded into the mesoderm layer of CAMs are quantified by the mean fluorescence. *P < 0.05, **P < 0.01 vs. cells without CCL28 and TGF-β; #P < 0.05, ##P < 0.001 vs. TGF-β–only–treated cells by 1-way ANOVA with multiple-comparisons test. (C) Expression levels and cellular localization of E-cadherin and β-catenin in Ca9.22 and YD10B OSCC cells treated with CCL28 and/or TGF-β. Representative immunofluorescence images. Scale bars: 100 μm. (D) Expression levels of E-cadherin, β-catenin, and EMT-regulating transcription factors in Ca9.22 and YD10B OSCC cells treated with CCL28 and/or TGF-β. (E) Cytosolic and nuclear β-catenin levels in Ca9.22 and YD10B OSCC cells treated with CCL28 and/or TGF-β. (D and E) Representative Western blot images.

Article Snippet: CCL28, RANKL, or OPG levels in cell culture media were measured with commercially available kits for CCL28 (BioLegend), RANKL (EIAab), or OPG (Boster) according to the manufacturer’s instructions.

Techniques: Fluorescence, Expressing, Immunofluorescence, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) Invasion of CCL28-knockdown OSCC cells. (B) Invasion of CCR3-knockdown OSCC cells. (C) Invasion of CCR10-knockdown OSCC cells. (A–C) OSCC cells were transduced with lentiviral particles with control shRNAs or 3 different shRNAs targeting CCL28, CCR10, or CCR3. Knockdown of CCL28, CCR10, or CCR3 in transduced cells was confirmed by Western blotting (top panels). Cell invasion is quantified as the number of invaded cells per field (mean ± SEM, n = 3). *P < 0.05, **P < 0.005 vs. control shRNA–transfected cells without CCL28; #P < 0.05, ##P < 0.01 vs. CCL28-, CCR3-, or CCR10-specific shRNA–transfected cells without CCL28 by 1-way ANOVA with multiple-comparisons test. (D) Invasion of CCL28- or CCR10-knockdown OSCC cells labeled with CFDA-SE and then suspended in a DMEM/Matrigel (4:1) mixture on the CAMs of fertilized eggs (mean ± SEM, n = 3). Representative images of CAM. Scale bars: 100 μm. Cells invaded into the mesoderm layer are quantified by the mean fluorescence. *P < 0.05 versus control shRNA–transfected cells without CCL28; #P < 0.01 vs. CCL28- or CCR10-knockdown cells without CCL28 by 1-way ANOVA with multiple-comparisons test. (E) CCL28, CCR3, or CCR10 mRNA levels in normal and HNSCC tissues. The data were obtained from the TCGA database. Box plots show the median and interquartile range. *P < 0.0001 vs. normal tissue by 2-tailed Student’s t test. (F) Kaplan-Meier survival curves for HNSCC patients with high or low expression of CCL28, CCR3, or CCR10 mRNA by the log-rank test.

Article Snippet: CCL28, RANKL, or OPG levels in cell culture media were measured with commercially available kits for CCL28 (BioLegend), RANKL (EIAab), or OPG (Boster) according to the manufacturer’s instructions.

Techniques: Knockdown, Transduction, Control, Western Blot, shRNA, Transfection, Labeling, Fluorescence, Expressing

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) Representative pathway reporter array (n = 2) for wild-type and CCR10-knockdown (KD) OSCC cells in the absence or presence of CCL28 (20 ng/mL). Reporter gene activities in CCL28-treated cells were normalized by those in untreated cells and represented as fold changes. (B) Correlations between CCL28 mRNA expression and RARβ mRNA expression in patients with HNSCC by Pearson’s correlation analysis. Scatter plots represent normalized RSEM values for each gene. (C) RARβ and RARβ2 expression in response to CCL28 treatment (20 pg/mL) in Ca9.22, YD10B, HSC2, or HSC3 OSCC cells. (D) RARβ and RARβ2 expression in CCL28-overexpressing or CCL28-knockdown Ca9.22 or YD10B OSCC cells. (E) RARβ expression in response to CCL28 treatment (20 pg/mL) in CCR3- or CCR10-downregulated Ca9.22 or YD10B OSCC cells. (C–E) Representative Western blot images. (F) Invasion of OSCC cells treated with the RARβ-selective antagonist LE135 or the inverse pan-RAR agonist BMS493 in the presence of CCL28 (20 pg/mL) (mean ± SEM, n = 3). *P < 0.05 and **P < 0.005 versus CCL28-untreated cells; #P < 0.05 and ##P < 0.01 versus CCL28-only-treated cells by 1-way ANOVA with multiple-comparisons test.

Article Snippet: CCL28, RANKL, or OPG levels in cell culture media were measured with commercially available kits for CCL28 (BioLegend), RANKL (EIAab), or OPG (Boster) according to the manufacturer’s instructions.

Techniques: Knockdown, Expressing, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) RARβ and RARβ2 expression levels in OSCC cells treated with CCL28 (20 pg/mL) and/or the selective RARα antagonist ER50891 (ER). (B) Invasion of OSCC cells treated with CCL28 (20 pg/mL) and/or the selective RARα antagonist ER50891 (ER) (mean ± SEM, n = 3). *P < 0.001 versus CCL28-untreated control cells; #P < 0.005 and ##P < 0.001 versus CCL28-only-treated cells by 1-way ANOVA with multiple-comparisons test. (C) Interaction between RARα and HDACs or DNMT in OSCC cells treated with CCL28 (20 pg/mL). Immune complexes were obtained using a Pierce Co-IP kit. (A and C) Representative Western blot images. (D) Acetylated histone H3 levels and HDAC1 interaction at the RARB promoter region of OSCC cells treated with CCL28 (20 pg/mL). Histone modification (H3K9ac) and HDAC1 binding were analyzed by ChIP-qPCR. Data are presented as the percentage of the total chromatin input (% input), and graphs are representative.

Article Snippet: CCL28, RANKL, or OPG levels in cell culture media were measured with commercially available kits for CCL28 (BioLegend), RANKL (EIAab), or OPG (Boster) according to the manufacturer’s instructions.

Techniques: Expressing, Control, Co-Immunoprecipitation Assay, Western Blot, Modification, Binding Assay, ChIP-qPCR

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) RANKL and OPG levels secreted by CCL28-treated OSCC cells into the culture media, and the RANKL/OPG ratio (mean ± SEM, n = 3). *P < 0.05 vs. CCL28-untreated cells by 2-tailed Student’s t test. (B) RANKL levels secreted by OSCC cells treated with the selective RARα antagonist ER50891 or the RARβ antagonist LE135 in the presence of CCL28 (mean ± SEM, n = 3). *P < 0.05 versus CCL28-untreated cells; #P < 0.05 versus CCL28-only-treated cells by 1-way ANOVA with multiple comparisons test. (C) RANKL and OPG levels secreted by CCL28-treated osteoblasts into the culture media, and the RANKL/OPG ratio (mean ± SEM, n = 3). *P < 0.05 and **P < 0.01 versus CCL28-untreated cells by 1-way ANOVA with multiple comparisons test. (D) Secreted levels of RANKL and OPG by CCL28-treated osteoblasts in the presence of conditioned media (CM) from OSCC cell lines, and the RANKL/OPG ratio (mean ± SEM, n = 3). #P < 0.05 and ##P < 0.01 versus control cells without CM; *P < 0.05 versus CM-only-treated cells by 1-way ANOVA with multiple-comparisons test. (E) Osteoclast formation in CCL28-treated BMMs in the presence of RANKL (mean ± SEM, n = 3). Representative images at ×100 original magnification. *P < 0.05 versus RANKL-only-treated cells by 1-way ANOVA with multiple comparisons test.

Article Snippet: CCL28, RANKL, or OPG levels in cell culture media were measured with commercially available kits for CCL28 (BioLegend), RANKL (EIAab), or OPG (Boster) according to the manufacturer’s instructions.

Techniques: Control

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: CCL28 was intraperitoneally administered to mice subcutaneously injected with Ca9.22 OSCC cells in the calvaria (n = 5 for control and n = 10 for experimental groups). (A) Tumor size (mean ± SEM). #P < 0.001 versus vehicle-treated mice by 1-way ANOVA with multiple comparisons test. (B) Representative CT 3D images of calvarial osteolytic lesions. (C) Bone morphometric parameters BV/TV and BS/TV (mean ± SEM). (D) Serum levels of bone turnover markers (mean ± SEM). (E) Representative images of H&E and TRAP staining in calvarial tissue sections. Scale bars: 100 μm. (F) Oc.S/BS determined from TRAP staining as the percentage of bone surface in contact with osteoclasts (mean ± SEM). (C, D, and F) #P < 0.05, ##P < 0.01, and ###P < 0.005 versus control mice; *P < 0.05 and **P < 0.01 versus OSCC cell–injected mice by 1-way ANOVA with multiple comparisons test. (G) Ki67, CD31, and RAR expression levels in calvarial tumor tissues of OSCC-injected mice. Left panel: Representative images of immunohistochemically stained tumor tissues. Scale bars: 100 μm. Graph shows quantified data. *P < 0.05 and **P < 0.01 versus OSCC cell–injected mice by 1-way ANOVA with multiple-comparisons test.

Article Snippet: CCL28, RANKL, or OPG levels in cell culture media were measured with commercially available kits for CCL28 (BioLegend), RANKL (EIAab), or OPG (Boster) according to the manufacturer’s instructions.

Techniques: Injection, Control, Staining, Expressing

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: CCL28 was intraperitoneally administered to mice injected with YD10B OSCC cells into the bone marrow of the right tibia (n = 5 for control and n = 7 for experimental groups). (A) Representative CT 3D images of osteolytic lesions in the tibia. (B) Bone morphometric parameters (mean ± SEM). (C) Serum levels of bone turnover markers (mean ± SEM). (D) Representative images of H&E and TRAP staining in tibial tissue sections. Scale bars: 100 μm. (E) Tumor area determined from H&E staining as the percentage of the total tumor area per tissue area. (F) Oc.S/BS determined from TRAP staining as the percentage of bone surface in contact with osteoclasts (mean ± SEM). (B, C, E, and F) #P < 0.05, ##P < 0.01, and ###P < 0.005 versus control mice; *P < 0.05 and **P < 0.01 versus OSCC cell–injected mice by 1-way ANOVA with multiple comparisons test. (G) Ki67, CD31, and RARβ expression levels in tibial tumor tissues of OSCC-injected mice. Left panel: Representative images of immunohistochemically stained tumor tissues. Scale bars: 100 μm. Right panel: Ki67-positive cells, CD31-positive vessels, and RARβ-positive cells were counted in tumor tissues. *P < 0.05 and **P < 0.01 versus OSCC cell–injected mice by 1-way ANOVA with multiple comparisons test.

Article Snippet: CCL28, RANKL, or OPG levels in cell culture media were measured with commercially available kits for CCL28 (BioLegend), RANKL (EIAab), or OPG (Boster) according to the manufacturer’s instructions.

Techniques: Injection, Control, Staining, Expressing

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) Representative images of IHC staining of CCL28, CCR3, CCR10, and RARβ in normal oral mucosa and OSCC tissues. Scale bars: 100 μm. Magnified images of the boxed area are shown in the insets. Scale bars: 20 μm. (B) Frequency of histoscores in normal oral mucosa and OSCC tissues. (C) Kaplan-Meier survival curve of patients with OSCC stratified based on CCL28, CCR3, CCR10, or RARβ expression by the log-rank test.

Article Snippet: CCL28, RANKL, or OPG levels in cell culture media were measured with commercially available kits for CCL28 (BioLegend), RANKL (EIAab), or OPG (Boster) according to the manufacturer’s instructions.

Techniques: Immunohistochemistry, Expressing