mouse anti jam3  (R&D Systems)

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: Sections were then blocked with 5% bovine serum albumin (BSA; Beijing Solarbio Science & Technology Co., Ltd.) for 20 min at room temperature to reduce non-specific binding, then incubated overnight at 4°C with primary antibodies against Ki-67 (cat. no. RMA-0731; Fuzhou Maixin Biotechnology Development Co., Ltd.), JAM3 (cat. no. bs-11086R; 1:200; rabbit; BIOSS), YAP1 (cat. no. 13584-1-AP; 1:500; rabbit; Proteintech Group, Inc.), E-Cadherin (cat. no. 20874-1-AP; 1:2,000; rabbit; Proteintech Group, Inc.), N-Cadherin (cat. no. 22018-1-AP; 1:2,000, rabbit; Proteintech Group, Inc.) and Vimentin (cat. no. 5741S; 1:600; rabbit; Cell Signaling Technology, Inc.).

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: Sections were then blocked with 5% bovine serum albumin (BSA; Beijing Solarbio Science & Technology Co., Ltd.) for 20 min at room temperature to reduce non-specific binding, then incubated overnight at 4°C with primary antibodies against Ki-67 (cat. no. RMA-0731; Fuzhou Maixin Biotechnology Development Co., Ltd.), JAM3 (cat. no. bs-11086R; 1:200; rabbit; BIOSS), YAP1 (cat. no. 13584-1-AP; 1:500; rabbit; Proteintech Group, Inc.), E-Cadherin (cat. no. 20874-1-AP; 1:2,000; rabbit; Proteintech Group, Inc.), N-Cadherin (cat. no. 22018-1-AP; 1:2,000, rabbit; Proteintech Group, Inc.) and Vimentin (cat. no. 5741S; 1:600; rabbit; Cell Signaling Technology, Inc.).

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: Sections were then blocked with 5% bovine serum albumin (BSA; Beijing Solarbio Science & Technology Co., Ltd.) for 20 min at room temperature to reduce non-specific binding, then incubated overnight at 4°C with primary antibodies against Ki-67 (cat. no. RMA-0731; Fuzhou Maixin Biotechnology Development Co., Ltd.), JAM3 (cat. no. bs-11086R; 1:200; rabbit; BIOSS), YAP1 (cat. no. 13584-1-AP; 1:500; rabbit; Proteintech Group, Inc.), E-Cadherin (cat. no. 20874-1-AP; 1:2,000; rabbit; Proteintech Group, Inc.), N-Cadherin (cat. no. 22018-1-AP; 1:2,000, rabbit; Proteintech Group, Inc.) and Vimentin (cat. no. 5741S; 1:600; rabbit; Cell Signaling Technology, Inc.).

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: Sections were then blocked with 5% bovine serum albumin (BSA; Beijing Solarbio Science & Technology Co., Ltd.) for 20 min at room temperature to reduce non-specific binding, then incubated overnight at 4°C with primary antibodies against Ki-67 (cat. no. RMA-0731; Fuzhou Maixin Biotechnology Development Co., Ltd.), JAM3 (cat. no. bs-11086R; 1:200; rabbit; BIOSS), YAP1 (cat. no. 13584-1-AP; 1:500; rabbit; Proteintech Group, Inc.), E-Cadherin (cat. no. 20874-1-AP; 1:2,000; rabbit; Proteintech Group, Inc.), N-Cadherin (cat. no. 22018-1-AP; 1:2,000, rabbit; Proteintech Group, Inc.) and Vimentin (cat. no. 5741S; 1:600; rabbit; Cell Signaling Technology, Inc.).

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: Sections were then blocked with 5% bovine serum albumin (BSA; Beijing Solarbio Science & Technology Co., Ltd.) for 20 min at room temperature to reduce non-specific binding, then incubated overnight at 4°C with primary antibodies against Ki-67 (cat. no. RMA-0731; Fuzhou Maixin Biotechnology Development Co., Ltd.), JAM3 (cat. no. bs-11086R; 1:200; rabbit; BIOSS), YAP1 (cat. no. 13584-1-AP; 1:500; rabbit; Proteintech Group, Inc.), E-Cadherin (cat. no. 20874-1-AP; 1:2,000; rabbit; Proteintech Group, Inc.), N-Cadherin (cat. no. 22018-1-AP; 1:2,000, rabbit; Proteintech Group, Inc.) and Vimentin (cat. no. 5741S; 1:600; rabbit; Cell Signaling Technology, Inc.).

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: Sections were then blocked with 5% bovine serum albumin (BSA; Beijing Solarbio Science & Technology Co., Ltd.) for 20 min at room temperature to reduce non-specific binding, then incubated overnight at 4°C with primary antibodies against Ki-67 (cat. no. RMA-0731; Fuzhou Maixin Biotechnology Development Co., Ltd.), JAM3 (cat. no. bs-11086R; 1:200; rabbit; BIOSS), YAP1 (cat. no. 13584-1-AP; 1:500; rabbit; Proteintech Group, Inc.), E-Cadherin (cat. no. 20874-1-AP; 1:2,000; rabbit; Proteintech Group, Inc.), N-Cadherin (cat. no. 22018-1-AP; 1:2,000, rabbit; Proteintech Group, Inc.) and Vimentin (cat. no. 5741S; 1:600; rabbit; Cell Signaling Technology, Inc.).

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: Sections were then blocked with 5% bovine serum albumin (BSA; Beijing Solarbio Science & Technology Co., Ltd.) for 20 min at room temperature to reduce non-specific binding, then incubated overnight at 4°C with primary antibodies against Ki-67 (cat. no. RMA-0731; Fuzhou Maixin Biotechnology Development Co., Ltd.), JAM3 (cat. no. bs-11086R; 1:200; rabbit; BIOSS), YAP1 (cat. no. 13584-1-AP; 1:500; rabbit; Proteintech Group, Inc.), E-Cadherin (cat. no. 20874-1-AP; 1:2,000; rabbit; Proteintech Group, Inc.), N-Cadherin (cat. no. 22018-1-AP; 1:2,000, rabbit; Proteintech Group, Inc.) and Vimentin (cat. no. 5741S; 1:600; rabbit; Cell Signaling Technology, Inc.).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Junctional Adhesion Molecule 3 Expression in the Mouse Airway Epithelium Is Linked to Multiciliated Cells

doi: 10.3389/fcell.2021.622515

Figure Lengend Snippet: Junctional adhesion molecule 3 (Jam3) is expressed in multiciliated cells in the mouse airway epithelium. (A) Immunohistochemistry of Jam3 in the mouse airway epithelium. Black arrows point to multiciliated cells, while black arrowheads point toward non-ciliated cells. (A’) A higher magnification image for a Jam3 immunohistochemistry in the mouse airway epithelium. (B’) A magnification of an immunohistochemistry image of Jam3 in the mouse airway epithelium. (B) Immunofluorescence in mouse whole-mount trachea for Jam3 in red (gray in panel B’ ), acetylated tubulin in green (gray in panel B’ ), and DAPI in blue (gray in panel B” ). (C) Confocal image with higher magnification for Jam3 localization in whole-mount tracheas, Jam3 in red (gray in panel C’ ), and acetylated tubulin in green (gray in panel C” ). (D,E) Jam3 immunofluorescence in MTECs differentiated for 14 days in vitro , nuclei in blue (gray in panels D’,E’ ), and Jam3 in green (gray in panels D’,E” ) using two different antibodies. Scale bar represents 10 μm in panel (C) , represents 10 μm, and represents 20 μm in panels (D,E) . White arrowhead point a MCC with low Jam3 expression levels.

Article Snippet: Whole-mount tracheas, MTECs in air liquid interface, or MTECs before reaching confluency were fixed in 4% PFA (PolyScience, Warrington, PA, United States) for 10 min at room temperature, permeabilized in PBS-Triton 0.1% for 15 min, and blocked in PBS-Triton with 2% bovine serum albumin (BSA) (Roche) for 1 h. Samples were incubated with primary antibodies anti-Jam3 (R&D Systems, #AF1213, 1:150), anti-Jam3 (Thermo Fisher, AB_2533486, 1:100), E-cadherin (BD Biosciences, #610182, 1:100), anti-ZO1 (1:100), Vangl1 (Sigma, HPA025235, 1:100), anti-FoxJ1 (Invitrogen, #14-9965-82, 1:200), AcTub (1;100), anti-Rab11a (Cell Signaling, Danvers, MA, United States, #2413S, 1:50) or anti-EEA1 (Cell Signaling, #3288S, 1:100), anti-Dnai1 (Thermo Fisher, PA554526, 1:100), anti-Daap1 (Sigma-Millipore, Burlington, MA, United States, HPA049468, 1:100), anti-centriolin (Santa Cruz, Santa Cruz, CA, United States, SE-365521, 1:100), and anti-alpha tubulin (Thermo Fisher, 32-2500, 1:100), diluted in PBS-Triton-2% BSA in a wet chamber overnight at 4°C.

Techniques: Immunohistochemistry, Immunofluorescence, In Vitro, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Junctional Adhesion Molecule 3 Expression in the Mouse Airway Epithelium Is Linked to Multiciliated Cells

doi: 10.3389/fcell.2021.622515

Figure Lengend Snippet: Junctional adhesion molecule 3 (Jam3) expression is restricted to a subset of multiciliated cells. (A,B) Representative images for Jam3 and Foxj1 (A) or acetylated tubulin (B) to evaluate the co-labeling of MCCs. Foxj1 in green (gray in panel A’ ), and Jam3 in red (gray in panel A” ) Acetylated tubulin in green (gray in panel B’ ), and Jam3 in red (gray in panel B” ) (C) Quantification of the number of cells that express Jam3 and acetylated tubulin or Jam3 and Foxj1. (D) Set of confocal images at different days of differentiation (from ALI 3 to ALI7) for Jam3 in red (gray in panel D” ), acetylated tubulin in green (gray in panel D’ ), and nuclei in blue. (E) Quantification of acetylated tubulin-positive cells with high levels of Jam3, low levels of Jam3, or negative for Jam3 from ALI4 to ALI7. No acetylated tubulin or Jam3-positive cells were detected in ALI3. (F) Relative gene expression levels for deuterosomal cells and mature ciliated cell markers in Jam3-positive or negative cells (all of them are Foxj1-positive cells). Scale bar in panels (A,B) represents 20 and 10 μm in panel (D) . Red arrow pointed Foxj1 positive cells which are Jam3 negative. Red starts marked Ac-tubulin positive cells which are Jam3 negative.

Article Snippet: Whole-mount tracheas, MTECs in air liquid interface, or MTECs before reaching confluency were fixed in 4% PFA (PolyScience, Warrington, PA, United States) for 10 min at room temperature, permeabilized in PBS-Triton 0.1% for 15 min, and blocked in PBS-Triton with 2% bovine serum albumin (BSA) (Roche) for 1 h. Samples were incubated with primary antibodies anti-Jam3 (R&D Systems, #AF1213, 1:150), anti-Jam3 (Thermo Fisher, AB_2533486, 1:100), E-cadherin (BD Biosciences, #610182, 1:100), anti-ZO1 (1:100), Vangl1 (Sigma, HPA025235, 1:100), anti-FoxJ1 (Invitrogen, #14-9965-82, 1:200), AcTub (1;100), anti-Rab11a (Cell Signaling, Danvers, MA, United States, #2413S, 1:50) or anti-EEA1 (Cell Signaling, #3288S, 1:100), anti-Dnai1 (Thermo Fisher, PA554526, 1:100), anti-Daap1 (Sigma-Millipore, Burlington, MA, United States, HPA049468, 1:100), anti-centriolin (Santa Cruz, Santa Cruz, CA, United States, SE-365521, 1:100), and anti-alpha tubulin (Thermo Fisher, 32-2500, 1:100), diluted in PBS-Triton-2% BSA in a wet chamber overnight at 4°C.

Techniques: Expressing, Labeling, Gene Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: Junctional Adhesion Molecule 3 Expression in the Mouse Airway Epithelium Is Linked to Multiciliated Cells

doi: 10.3389/fcell.2021.622515

Figure Lengend Snippet: Junctional adhesion molecule 3 (Jam3) localizes at cell–cell contacts and in apical sorting endosomes. (A) Confocal Z-planes from apical to more basal ones (Z3 to Z5) of Jam3 in MCCs. White arrows point to Jam3 localization at cell contacts, and white arrowheads denoted Jam3 localization in apically located endosomes. Jam3 in red (gray in panel A’), and nucleus in blue (gray in panel A”). (B) Serial confocal Z-planes (Z3 to Z5) of Jam3 co-localization with ZO1 in MCCs. Jam3 in green (gray in panel B’), and ZO1 in red (gray in panel B”). (C) Immunofluorescence in mouse whole-mount trachea for Jam3 in red (gray in panel C’ ) and nuclei in blue. (D) A zoom for a region of interest from panel (A) showing Jam3 localization in a group of cells. (E) A single confocal plane image for Jam3 in red (gray in panel E” ) and EEA1 in green (gray in panel E”’ ) co-localization analyses (white in panel E’ ). White arrows point to Jam3 and EEA1 co-localization in endosomes, and white arrowheads denoted Jam3 endosomes which are EEA negative. Note that not all EEA1-positive endosomes are positive for Jam3. The step size between Z planes is 1 μm. Scale bar in panels ( A,B,D ) represents 10 μm. The dotted white box represents the ROI depicted in panel (D) .

Article Snippet: Whole-mount tracheas, MTECs in air liquid interface, or MTECs before reaching confluency were fixed in 4% PFA (PolyScience, Warrington, PA, United States) for 10 min at room temperature, permeabilized in PBS-Triton 0.1% for 15 min, and blocked in PBS-Triton with 2% bovine serum albumin (BSA) (Roche) for 1 h. Samples were incubated with primary antibodies anti-Jam3 (R&D Systems, #AF1213, 1:150), anti-Jam3 (Thermo Fisher, AB_2533486, 1:100), E-cadherin (BD Biosciences, #610182, 1:100), anti-ZO1 (1:100), Vangl1 (Sigma, HPA025235, 1:100), anti-FoxJ1 (Invitrogen, #14-9965-82, 1:200), AcTub (1;100), anti-Rab11a (Cell Signaling, Danvers, MA, United States, #2413S, 1:50) or anti-EEA1 (Cell Signaling, #3288S, 1:100), anti-Dnai1 (Thermo Fisher, PA554526, 1:100), anti-Daap1 (Sigma-Millipore, Burlington, MA, United States, HPA049468, 1:100), anti-centriolin (Santa Cruz, Santa Cruz, CA, United States, SE-365521, 1:100), and anti-alpha tubulin (Thermo Fisher, 32-2500, 1:100), diluted in PBS-Triton-2% BSA in a wet chamber overnight at 4°C.

Techniques: Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Junctional Adhesion Molecule 3 Expression in the Mouse Airway Epithelium Is Linked to Multiciliated Cells

doi: 10.3389/fcell.2021.622515

Figure Lengend Snippet: Downregulation of Jam3 does not alter cilia structure and function. (A–C) Mean mRNA expression levels of Jam3 (A) , Jam1 (B) , and Jam2 (C) assessed in differentiated MTECs infected with control viruses (Luc KD) or Jam3-shRNAs (Jam3 KD), n = 3. (D,E) Scanning electron micrographs of Jam3-KD and control (Luc KD) MTECs differentiated for 14 days. (F,G) Lateral and top views of confocal images for acetylated tubulin (in green), ZO-1 (in red), and nucleus (in blue) in Jam3-KD (G,G’) and Luc KD (F,F’) MTECs. (H) Cilia beating frequency quantification as number of beats per second in cells treated with Luc KD and Jam3 KD MTECs. (I,J) Basal body staining in control (I) and Jam3 KD cells (J) . (K,L) Black and white image obtained to calculate the Mm ratio in a control cell (K) and a Jam3 KD cell (L) . (M) Mm ratio quantification for individual cells. n > 60 in Control and Jam3 KD conditions. (N,O) Centriolin staining in control (Luc KD) and Jam3-KD cells in ALI 5. (P) Quantification of MCCs in different stages of differentiation in control and Jam3 KD cells. Type II/III are those cells with Centriolin staining in aggregates (pink arrowheads) while Type IV/V are those cells with Centriolin staining dispersed at the apical membrane (white arrowheads). Scale bar in panels (F,G) represent 20 μm. p -values in all conditions were obtained using t -test.

Article Snippet: Whole-mount tracheas, MTECs in air liquid interface, or MTECs before reaching confluency were fixed in 4% PFA (PolyScience, Warrington, PA, United States) for 10 min at room temperature, permeabilized in PBS-Triton 0.1% for 15 min, and blocked in PBS-Triton with 2% bovine serum albumin (BSA) (Roche) for 1 h. Samples were incubated with primary antibodies anti-Jam3 (R&D Systems, #AF1213, 1:150), anti-Jam3 (Thermo Fisher, AB_2533486, 1:100), E-cadherin (BD Biosciences, #610182, 1:100), anti-ZO1 (1:100), Vangl1 (Sigma, HPA025235, 1:100), anti-FoxJ1 (Invitrogen, #14-9965-82, 1:200), AcTub (1;100), anti-Rab11a (Cell Signaling, Danvers, MA, United States, #2413S, 1:50) or anti-EEA1 (Cell Signaling, #3288S, 1:100), anti-Dnai1 (Thermo Fisher, PA554526, 1:100), anti-Daap1 (Sigma-Millipore, Burlington, MA, United States, HPA049468, 1:100), anti-centriolin (Santa Cruz, Santa Cruz, CA, United States, SE-365521, 1:100), and anti-alpha tubulin (Thermo Fisher, 32-2500, 1:100), diluted in PBS-Triton-2% BSA in a wet chamber overnight at 4°C.

Techniques: Expressing, Infection, Control, Staining, Membrane

Journal: Frontiers in Cell and Developmental Biology

Article Title: Junctional Adhesion Molecule 3 Expression in the Mouse Airway Epithelium Is Linked to Multiciliated Cells

doi: 10.3389/fcell.2021.622515

Figure Lengend Snippet: Downregulation of Jam3 expression does not affect epithelial integrity but delays airway epithelial monolayer TEER during expansion. (A) Confocal images corresponding to orthogonal views in MTECs control (A) and Jam3 KD cells (B) . Streptavidin Alexa 555 (in red) was only observed at the apical membrane. Phalloidin (in green) was used to label the cortical actin in both the apical and basolateral surfaces and Dapi (in blue) for nucleus. (C,D) Confocal images to evaluate ZO-1 recruitment to the junction in Luc KD (C) and Jam3 KD (D) cells. (E,F) Cell size (E) and cell–cell contact number (F) evaluation in confocal images of ZO-1 in Luc KD and Jam3 KD cells. (G) Transepithelial resistance (TEER) measure was used to test tight-junction permeability during the differentiation process of MTECs in Jam3-KD compared to Luc-KD cells. Scale bar in panels (A–D) represents 20 μm. p -values in all conditions were obtained using t -test.

Article Snippet: Whole-mount tracheas, MTECs in air liquid interface, or MTECs before reaching confluency were fixed in 4% PFA (PolyScience, Warrington, PA, United States) for 10 min at room temperature, permeabilized in PBS-Triton 0.1% for 15 min, and blocked in PBS-Triton with 2% bovine serum albumin (BSA) (Roche) for 1 h. Samples were incubated with primary antibodies anti-Jam3 (R&D Systems, #AF1213, 1:150), anti-Jam3 (Thermo Fisher, AB_2533486, 1:100), E-cadherin (BD Biosciences, #610182, 1:100), anti-ZO1 (1:100), Vangl1 (Sigma, HPA025235, 1:100), anti-FoxJ1 (Invitrogen, #14-9965-82, 1:200), AcTub (1;100), anti-Rab11a (Cell Signaling, Danvers, MA, United States, #2413S, 1:50) or anti-EEA1 (Cell Signaling, #3288S, 1:100), anti-Dnai1 (Thermo Fisher, PA554526, 1:100), anti-Daap1 (Sigma-Millipore, Burlington, MA, United States, HPA049468, 1:100), anti-centriolin (Santa Cruz, Santa Cruz, CA, United States, SE-365521, 1:100), and anti-alpha tubulin (Thermo Fisher, 32-2500, 1:100), diluted in PBS-Triton-2% BSA in a wet chamber overnight at 4°C.

Techniques: Expressing, Control, Membrane, Permeability

Journal: Frontiers in Cell and Developmental Biology

Article Title: Junctional Adhesion Molecule 3 Expression in the Mouse Airway Epithelium Is Linked to Multiciliated Cells

doi: 10.3389/fcell.2021.622515

Figure Lengend Snippet: Junctional adhesion molecule 3 (Jam 3) expression varies along the differentiation process but does not affect cell differentiation. (A–C) Quantification of mRNA expression of Krt5 (basal marker, in panel A ), FoxJ1 (multiciliated cells marker in panel B ), and Scgb1a1 (secretory cells marker in panel C ) expression in ALI 0, ALI 4, ALI 7, and ALI14. (D–F) Quantification of mRNA expression of Jam1 (E) , Jam2 (F) , and Jam3 (D) expression in ALI 0, ALI 4, ALI 7, and ALI14. Mean relative to control cells and standard deviation as error bars were plotted for each lineage marker, n = 4. (G) Jam3 immunofluorescence in BSCs during expansion in vitro , nucleus in blue (gray in panel G’ ) and Jam3 in red (gray in panel G” ). (H–J) mRNA expression levels of Krt5 (J) , FoxJ1 (H) , and Scgb1a1 (I) in Luc KD (C) and Jam3 KD (D) MTECs. Mean relative to control cells and standard deviation as error bars were plotted for each lineage marker, n = 4. (K,L) Confocal images for Foxj1 (green in panels K,L and gray in panels K’,L’ ) immunofluorescence in Luc KD (K) or Jam3 KD (L) cells in ALI14. (M) Relative quantification of Foxj1-positive cells along the differentiation process in Luc KD and Jam3 KD cells from ALI 4 to ALI 17. (N–S) Mean mRNA expression levels of Jam3 (O) , Jam1 (P) , Jam2 (N) , Krt5 (D) , Foxj1 (E) , and Scgb1a1 (F) assessed in differentiated MTECs infected with control viruses (Luc KD), Jam2-shRNAs (Jam2 KD), and double knockdown (Jam2 and Jam3), n = 4. Scale bar in panels (G,K,L) represents 20 μm. p -values in all conditions were obtained using the t -test. * p < 0.05.

Article Snippet: Whole-mount tracheas, MTECs in air liquid interface, or MTECs before reaching confluency were fixed in 4% PFA (PolyScience, Warrington, PA, United States) for 10 min at room temperature, permeabilized in PBS-Triton 0.1% for 15 min, and blocked in PBS-Triton with 2% bovine serum albumin (BSA) (Roche) for 1 h. Samples were incubated with primary antibodies anti-Jam3 (R&D Systems, #AF1213, 1:150), anti-Jam3 (Thermo Fisher, AB_2533486, 1:100), E-cadherin (BD Biosciences, #610182, 1:100), anti-ZO1 (1:100), Vangl1 (Sigma, HPA025235, 1:100), anti-FoxJ1 (Invitrogen, #14-9965-82, 1:200), AcTub (1;100), anti-Rab11a (Cell Signaling, Danvers, MA, United States, #2413S, 1:50) or anti-EEA1 (Cell Signaling, #3288S, 1:100), anti-Dnai1 (Thermo Fisher, PA554526, 1:100), anti-Daap1 (Sigma-Millipore, Burlington, MA, United States, HPA049468, 1:100), anti-centriolin (Santa Cruz, Santa Cruz, CA, United States, SE-365521, 1:100), and anti-alpha tubulin (Thermo Fisher, 32-2500, 1:100), diluted in PBS-Triton-2% BSA in a wet chamber overnight at 4°C.

Techniques: Expressing, Cell Differentiation, Marker, Control, Standard Deviation, Immunofluorescence, In Vitro, Quantitative Proteomics, Infection, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: Junctional Adhesion Molecule 3 Expression in the Mouse Airway Epithelium Is Linked to Multiciliated Cells

doi: 10.3389/fcell.2021.622515

Figure Lengend Snippet: Junctional adhesion molecule 3 (Jam 3) expression is enhanced in MTECs treated with IL6 during differentiation. (A–F) mRNA expression levels of Krt5 for basal cells (A) , Foxj1 for MCCs (B) , and Scgb1a1 for club cells (C) , Jam1 (D) , Jam3 (E) , and Jam2 (F) in MTECs treated with DMSO or DAPT for 14 days. (G–L) mRNA expression levels of Krt5 for basal cells (G) , Foxj1 for MCCs (H) , and Scgb1a1 for club cells (H) , Jam1 (I) , Jam3 (K) , and Jam2 (L) in MTECs treated with PBS or IL6 for 14 days. Mean expression values relative to DMSO-treated cells and standard deviation as error bars were plotted for each lineage marker, n = 4. (M–N) Jam3 localization in MTECs treated for 14 days with PBS (M) or IL6 (N) . The cell membrane was labeled using actin in green (gray in panels M’,N’ ) and Jam3 in red (gray in panels M”,N” ). (O–Q) Jam3 localization in MTECs treated for 1 h with PBS (O) or two concentrations of histamine (P,Q) . Scale bar in panels (M–Q) represents 20 μm. p -values in all conditions were obtained using the t -test.

Article Snippet: Whole-mount tracheas, MTECs in air liquid interface, or MTECs before reaching confluency were fixed in 4% PFA (PolyScience, Warrington, PA, United States) for 10 min at room temperature, permeabilized in PBS-Triton 0.1% for 15 min, and blocked in PBS-Triton with 2% bovine serum albumin (BSA) (Roche) for 1 h. Samples were incubated with primary antibodies anti-Jam3 (R&D Systems, #AF1213, 1:150), anti-Jam3 (Thermo Fisher, AB_2533486, 1:100), E-cadherin (BD Biosciences, #610182, 1:100), anti-ZO1 (1:100), Vangl1 (Sigma, HPA025235, 1:100), anti-FoxJ1 (Invitrogen, #14-9965-82, 1:200), AcTub (1;100), anti-Rab11a (Cell Signaling, Danvers, MA, United States, #2413S, 1:50) or anti-EEA1 (Cell Signaling, #3288S, 1:100), anti-Dnai1 (Thermo Fisher, PA554526, 1:100), anti-Daap1 (Sigma-Millipore, Burlington, MA, United States, HPA049468, 1:100), anti-centriolin (Santa Cruz, Santa Cruz, CA, United States, SE-365521, 1:100), and anti-alpha tubulin (Thermo Fisher, 32-2500, 1:100), diluted in PBS-Triton-2% BSA in a wet chamber overnight at 4°C.

Techniques: Expressing, Standard Deviation, Marker, Membrane, Labeling