Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: JAM3 is downregulated in LSCC tissues and samples at both mRNA and protein levels. (A) GSE216664, GSE59102 and GSE51985 were used to analyze the different expression levels of JAM3 between N and LSCC T tissues. For GSE216664 and GSE51985, N and T tissues were from the same LSCC patients; for GSE59102, N tissues were ANM tissues from different patients with LSCC. Data are presented as the mean with 95% CI indicated by error bars. **P<0.01, ***P<0.001, ****P<0.0001. (B) Protein levels of JAM3 in the Clinical Proteomic Tumor Analysis Consortium and the International Cancer Proteogenome Consortium datasets among different grades of HNSCC tissues. Data are presented as the mean and range. *P<0.05, **P<0.01, ****P<0.0001 vs. Grade 3. (C) Representative images of JAM3 IHC staining of LSCC tissues and paired ANM tissues. Red scale bar, 150 µm; Black scale bar, 20 µm. (D) IHC results of clinical tissues were analyzed by paired two-tailed Student's t-test. Data are presented as the mean with 95% CI. ****P<0.0001. (E) mRNA expression levels of JAM3 in HaCaT, AMC-HN-8, FD-LSC-1, FaDu and HN30 cell lines. Data are presented as the mean ± SD of three independent experiments. ****P<0.0001 vs. HaCaT. ANM, adjacent normal mucosa; IHC, immunohistochemistry; JAM3, junctional adhesion molecule 3; LSCC, laryngeal squamous cell carcinoma; N, non-tumor; T, tumor.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques: Expressing, Immunohistochemistry, Two Tailed Test

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: Aberrant hypermethylation in the JAM3 promoter is related to low expression of JAM3 . (A) Analysis of JAM3 expression and methylation levels, as well as their correlation using the Gene Expression Omnibus datasets GSE33202 and GSE33205. β value indicates the methylation level of the CpG site. Data are presented as the mean with 95% CI. ***P<0.001, ****P<0.0001. (B) Analysis of JAM3 expression and methylation levels, as well as their correlation in The Cancer Genome Atlas HNSCC samples. β value indicates the methylation level of the CpG site. Data are presented as the mean and range. *P<0.05, ****P<0.0001. (C) Kaplan-Meier plots, generated using the MethSurv webtool highlighted the relationship between methylation levels at certain JAM3 sites and overall survival rates in patients with HNSCC. (D) CpG island (blue area) predicted by MethPrimer tool were detected by bisulfite sequencing PCR in AMC-HN-8 and FD-LSC-1 cell lines. Each row represents an individual cloned allele. Black circles show methylated CpG sites and white circles show unmethylated CpG sites. The percentage methylation rate of each CpG site is shown by the blue-yellow columns; blue indicates unmethylated and yellow indicates methylated sites. (E) Reverse transcription-quantitative PCR and western blotting showing restored expression of JAM3 in both AMC-HN-8 and FD-LSC-1 cells treated with 5-Aza (5 µM) for 72 h. Data are presented as the mean ± SD of three independent experiments. **P<0.01, ***P<0.001 vs. the 5-Aza-group. 5-Aza, 5-Aza-2′-deoxycytidine; HNSCC, head and neck squamous cell carcinoma; JAM3, junctional adhesion molecule 3; FPKM, fragments per kilobase million; LR test, log-likelihood ratio test; HR, hazard ratio.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques: Expressing, Methylation, Generated, Methylation Sequencing, Clone Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: Overexpression of JAM3 suppresses laryngeal squamous cell carcinoma cell proliferation, migration and invasion. (A) AMC-HN-8 and FD-LSC-1 cells were transfected with JAM3 overexpression plasmid or Vec. Expression of JAM3 was examined by reverse transcription-quantitative PCR. Cell proliferation was detected by (B) Cell Counting Kit 8 and (C) colony formation assays. (D) Migration and (E) invasion abilities of AMC-HN-8 and FD-LSC-1 cells after transfection were determined by Transwell assays. Scale bar, 200 µm. Data are presented as the mean ± SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. Vec. JAM3, junctional adhesion molecule 3; Vec, p3×Flag-CMV-10 empty vector.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques: Over Expression, Migration, Transfection, Plasmid Preparation, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Counting

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: Knockdown of JAM3 promotes laryngeal squamous cell carcinoma cell proliferation, migration and invasion. (A) AMC-HN-8 and FD-LSC-1 cells were transfected with siRNAs targeting JAM3 (si-JAM3-1, si-JAM3-2) or si-NC. Expression of JAM3 was examined by reverse transcription-quantitative PCR. Cell proliferation was detected by (B) Cell Counting Kit 8 and (C) colony formation assays. (D) Migration and (E) invasion abilities of AMC-HN-8 and FD-LSC-1 cells after transfection were determined by Transwell assays. Scale bar, 200 µm. Data are presented as the mean ± SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. the si-NC group. JAM3, junctional adhesion molecule 3; NC, negative control; si, small interfering.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques: Knockdown, Migration, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Counting, Negative Control

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: JAM3 mediates laryngeal squamous cell carcinoma tumorigenesis through the Hippo pathway. (A) Protein levels of Flag, JAM3, p-LATS1 (Thr1079), LATS1, p-YAP1 (Ser127), YAP1 and β-actin in AMC-HN-8 and FD-LSC-1 cells with overexpression or knockdown of JAM3 were detected by western blotting. (B) Expression of YAP1 in AMC-HN-8 and FD-LSC-1 cells after transfection with the JAM3 overexpression plasmid or si-JAM3 were detected by confocal microscopy. Scale bar, 50 µm. Fluorescence ratio of YAP1 in the cytoplasm and nucleus of (C) AMC-HN-8 and (D) FD-LSC-1 cells was calculated by ImageJ and analyzed by two-tailed Student's t-test or one-way ANOVA. Data are presented as the mean ± SD of three independent experiments. *P<0.05 vs. Vec; # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001 vs. si-NC group. JAM3, junctional adhesion molecule 3; LATS1, large tumor suppressor kinase 1; NC, negative control; p-, phosphorylated; si, small interfering; Vec, p3×Flag-CMV-10 empty vector; YAP1, yes-associated protein 1.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques: Over Expression, Knockdown, Western Blot, Expressing, Transfection, Plasmid Preparation, Confocal Microscopy, Fluorescence, Two Tailed Test, Negative Control

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: Knockdown of JAM3 promotes tumorigenicity of laryngeal squamous cell carcinoma cells in vivo . (A) Representative images of tumors in nude mice after subcutaneous injection of AMC-HN-8 cells transfected with si-NC and si-JAM3. (B) Tumor growth curve was plotted using xenograft tumor volume data. ***P<0.001 vs. si-NC. (C) Tumor weight was measured after tumor excision. *P<0.05 vs. si-NC group. (D) Relative expression levels of JAM3 in xenograft tumors as determined by reverse transcription-quantitative PCR analysis. Data are presented as the mean ± SD of three independent experiments. ****P<0.0001 vs. si-NC. (E) Hematoxylin and eosin staining of xenograft tumors. Immunohistochemistry staining of (F) JAM3, Ki-67 and YAP1, and (G) E-cadherin, N-cadherin and Vimentin in xenograft tumors. Scale bar, 20 µm. JAM3, junctional adhesion molecule 3; NC, negative control; si, small interfering; YAP1, yes-associated protein 1.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques: Knockdown, In Vivo, Injection, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Staining, Immunohistochemistry, Negative Control

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: Mechanism by which epigenetic silencing of JAM3 promotes LSCC development by inhibiting the Hippo pathway. JAM3, junctional adhesion molecule 3; LATS1, large tumor suppressor kinase 1; YAP1, yes-associated protein 1; p, phosphorylated; EMT, epithelial-mesenchymal transition.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques: