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

Journal: Clinical Epigenetics

Article Title: Epigenetic silencing of JAM3 promotes esophageal cancer development by activating Wnt signaling

doi: 10.1186/s13148-022-01388-3

Figure Lengend Snippet: The effect of JAM3 on cell cycle and apoptosis. a The effect of JAM3 on cell phase in EC cells. The bar diagram represents the percentage. b The effect of JAM3 on apoptosis in EC cells. Each experiment was repeated in triplicate. * p < 0.05. ** p < 0.01. *** p < 0.001. c Western blot shows the levels of JAM3, cyclinD1, cyclin E1, cyclin A2, BAX, Bcl2, caspase3 and cleaved-caspase3. Actin: internal control. Scrambled: siRNA negative control; siRNA#2: siRNA targeting JAM3

Article Snippet: Antibodies were diluted according to the manufacturer’s instructions, including JAM3 (Abcam, USA), Caspase3/Cleaved-caspase3 (Proteintech, USA), MMP2 (Proteintech, USA), MMP9 (Proteintech, USA), cyclinD1 (Proteintech, USA), cyclin A (Proteintech, USA), cyclin E1 (Proteintech, USA), Myc (Proteintech, USA), β-catenin (Proteintech, USA), p-β-catenin (ZENBIO, China) and Actin (Proteintech, USA).

Techniques: Western Blot, Control, Negative Control

Journal: Clinical Epigenetics

Article Title: Epigenetic silencing of JAM3 promotes esophageal cancer development by activating Wnt signaling

doi: 10.1186/s13148-022-01388-3

Figure Lengend Snippet: The effect of JAM3 on Wnt/β-catenin signaling in EC. a Western blot shows the levels of JAM3, β-catenin, p-β-catenin, Myc and cyclinD1 in EC cells. Actin: internal control. Scrambled: siRNA negative control; siRNA#2: siRNA targeting JAM3. b, c Representative tumors from JAM3 unexpressed and re-expressed KYSE410 cell xenografts in mice. d Growth curves of JAM3 unexpressed and re-expressed KYSE410 cell tumors. *** p < 0.001. e Tumor weight in JAM3 unexpressed and re-expressed KYSE410 cell xenografts. Bars represent the mean of tumor weight. *** p < 0.001. f Representative results of immunohistochemistry for JAM3, β-catenin and p-β-catenin in xenografts

Article Snippet: Antibodies were diluted according to the manufacturer’s instructions, including JAM3 (Abcam, USA), Caspase3/Cleaved-caspase3 (Proteintech, USA), MMP2 (Proteintech, USA), MMP9 (Proteintech, USA), cyclinD1 (Proteintech, USA), cyclin A (Proteintech, USA), cyclin E1 (Proteintech, USA), Myc (Proteintech, USA), β-catenin (Proteintech, USA), p-β-catenin (ZENBIO, China) and Actin (Proteintech, USA).

Techniques: Western Blot, Control, Negative Control, Immunohistochemistry

Journal: Clinical Epigenetics

Article Title: Epigenetic silencing of JAM3 promotes esophageal cancer development by activating Wnt signaling

doi: 10.1186/s13148-022-01388-3

Figure Lengend Snippet: The effect of JAM3 on cell cycle and apoptosis. a The effect of JAM3 on cell phase in EC cells. The bar diagram represents the percentage. b The effect of JAM3 on apoptosis in EC cells. Each experiment was repeated in triplicate. * p < 0.05. ** p < 0.01. *** p < 0.001. c Western blot shows the levels of JAM3, cyclinD1, cyclin E1, cyclin A2, BAX, Bcl2, caspase3 and cleaved-caspase3. Actin: internal control. Scrambled: siRNA negative control; siRNA#2: siRNA targeting JAM3

Article Snippet: Antibodies were diluted according to the manufacturer’s instructions, including JAM3 (Abcam, USA), Caspase3/Cleaved-caspase3 (Proteintech, USA), MMP2 (Proteintech, USA), MMP9 (Proteintech, USA), cyclinD1 (Proteintech, USA), cyclin A (Proteintech, USA), cyclin E1 (Proteintech, USA), Myc (Proteintech, USA), β-catenin (Proteintech, USA), p-β-catenin (ZENBIO, China) and Actin (Proteintech, USA).

Techniques: Western Blot, Control, Negative Control

Journal: Clinical Epigenetics

Article Title: Epigenetic silencing of JAM3 promotes esophageal cancer development by activating Wnt signaling

doi: 10.1186/s13148-022-01388-3

Figure Lengend Snippet: The effect of JAM3 on cell proliferation, invasion and migration. a Growth curves represent the effects of JAM3 in EC cells. b Colony formation results show that the colony number was reduced by re-expression of JAM3 in KYSE30 and KYSE410 cells, and increased by knockdown of JAM3 in YES2 and KYSE450 cells. The average number of tumor clones is represented by the bar diagram. c Cell migration experiment in KYSE30 and KYSE410 cells before and after re-expression of JAM3, and in YES2 and KYSE450 cells before and after knockdown of JAM3. The average number of migration cells is presented by a bar diagram. d Cell invasion experiment in KYSE30 and KYSE410 cells before and after re-expression of JAM3, and in YES2 and KYSE450 cells before and after knockdown of JAM3. The average number of invasion cells is presented by a bar diagram. Each experiment was repeated in triplicate. * p < 0.05. ** p < 0.01. *** p < 0.001. e Western blots show the effects of knockdown of JAM3 by different siRNA. Scrambled: siRNA negative control; siRNA#1 and siRNA#2: siRNA for JAM3. f The levels of JAM3, MMP2 and MMP9 were detected by western blot. Actin: internal control. Scrambled: siRNA negative control; siRNA#2: siRNA targeting JAM3

Article Snippet: Antibodies were diluted according to the manufacturer’s instructions, including JAM3 (Abcam, USA), Caspase3/Cleaved-caspase3 (Proteintech, USA), MMP2 (Proteintech, USA), MMP9 (Proteintech, USA), cyclinD1 (Proteintech, USA), cyclin A (Proteintech, USA), cyclin E1 (Proteintech, USA), Myc (Proteintech, USA), β-catenin (Proteintech, USA), p-β-catenin (ZENBIO, China) and Actin (Proteintech, USA).

Techniques: Migration, Expressing, Knockdown, Clone Assay, Western Blot, Negative Control, Control