Journal: Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/β-catenin/CCND1 signaling

doi: 10.1172/jci93198

Figure Lengend Snippet: Figure 2. JAM3 promotes the self-renewal of LICs through enhanced cell cycle entry. (A) Representative flow cytometric analysis for WT and Jam3-null L-GMP cells of the recipients upon the secondary transplantation. (B) Quantification of frequencies of L-GMP cells in A (n = 3; ***P < 0.001, Student’s t test). (C and D) Survival data for recipient mice receiving WT or Jam3-null L-GMP cells upon the second to third transplantation (n = 5; **P < 0.01, log-rank test). (E–G) Representative images of colony formation of WT and Jam3-null YFP+Mac-1+c-Kit+ LICs of the secondary recipients in the first plating (E). The colony numbers (F) and total cell numbers of colonies in E (G) were counted (n = 3; ***P < 0.001, Student’s t test). (H–J) Representative images of colony formation of WT and Jam3-null leukemia cells clonogenically derived from the first plating (H). The colony numbers (I) and total cell numbers of colonies in H (J) were calculated (n = 3; ***P < 0.001, Student’s t test). (K) Cell cycle status was determined in WT and Jam3-null YFP+Mac-1+c-Kit+ LICs of the secondary recipients. (L) Quantitative analysis of the cell cycle distribution in K (n = 4–6; ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). (M) CFSE-labeled WT and Jam3-null leukemia cells of secondary recip- ients were transplanted and analyzed for the homed CFSE+ cells in the recipients’ BM and spleen (n = 5–6). (N) WT and Jam3-null leukemia cells of secondary recipients were transplanted into the recipient mice by intratibial injection, followed by the examination of leukemia cells 2 weeks later (n = 5; ***P < 0.001, Student’s t test). (O) Representative flow cytometric analysis of apoptosis of WT or Jam3-null YFP+Mac-1+c-Kit+ LICs. (P) Quantification of data in O (n = 4). Experiments were conducted 3–5 times for validation.

Article Snippet: Right after transplantation, 100 μg functional anti-JAM3 antibodies (catalog MCA5935XZ; clone CRAM-18 F26, Bio-Rad) (39, 40) or PBS were delivered into recipient mice via i.p. injection.

Techniques: Transplantation Assay, Derivative Assay, Labeling, Injection, Biomarker Discovery

Journal: Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/β-catenin/CCND1 signaling

doi: 10.1172/jci93198

Figure Lengend Snippet: Figure 3. JAM3 maintains the CCND1 level to promote the self-renewal of LICs. (A and B) GO (biological process) and KEGG (pathway) analyses of the microarray data of WT or Jam3-null YFP+Mac-1+c-Kit+ LICs. Candidate changes are highlighted in red. (C) Potential candidates related to self-renewal, cell cycle, and Wnt signaling were examined in WT and Jam3-null LICs by quantitative RT-PCR (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test). (D) CCND1 levels were compared between WT and Jam3-null YFP+Mac-1+c-Kit+ LICs by immunoblotting. (E) Ccnd1 was ectopically expressed in Jam3-null leukemia cells and injected into recipient mice. Survival was compared among the mice receiving WT cells, Jam3-null cells, and Ccnd1-overexpressing WT or Jam3-null cells (n = 5–6; ***P < 0.001, log-rank test). (F) CCND1 levels were validated in leukemia cells from the rescue experiment in E. (G) The cell cycle distribution in YFP+Mac-1+c-Kit+ LICs from the rescue experiment in E was determined using Ki-67 and Hoechst 33342 staining (n = 3–5; *P < 0.05, **P < 0.01, 2-way ANOVA followed by Bonferroni’s post-test). Experiments were conducted 3–5 times for validation.

Article Snippet: Right after transplantation, 100 μg functional anti-JAM3 antibodies (catalog MCA5935XZ; clone CRAM-18 F26, Bio-Rad) (39, 40) or PBS were delivered into recipient mice via i.p. injection.

Techniques: Microarray, Quantitative RT-PCR, Western Blot, Injection, Staining, Biomarker Discovery

Journal: Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/β-catenin/CCND1 signaling

doi: 10.1172/jci93198

Figure Lengend Snippet: Figure 4. JAM3 collaborates with LRP5 to activate β-catenin/CCND1 signaling. (A) Phospho–β-catenin (S552) and total β-catenin levels were evaluated between WT and Jam3-null YFP+Mac-1+c-Kit+ LICs by immunoblotting. (B) β-Catenin levels were compared between WT and Jam3-null YFP+Mac-1+c-Kit+ LICs by immunofluorescence staining. Scale bars: 5 µm. (C) A constitutively active form of phospho–β-catenin (S37A, β-cateninCN) was subcloned in the pCDH- EF1a-T2A-mCherry vector and ectopically expressed in Jam3-null leukemia cells, which were then injected into recipient mice. Survival was compared among the mice receiving WT cells, Jam3-null cells, and β-cateninCN–overexpressing WT or Jam3-null cells (n = 5–6; *P < 0.05, **P < 0.01, log-rank test). (D) Phos- pho–β-catenin (S552) and total β-catenin levels were validated in leukemia cells from the rescue experiment in C. (E) The cell cycle distribution in YFP+Mac-1+

Article Snippet: Right after transplantation, 100 μg functional anti-JAM3 antibodies (catalog MCA5935XZ; clone CRAM-18 F26, Bio-Rad) (39, 40) or PBS were delivered into recipient mice via i.p. injection.

Techniques: Western Blot, Immunofluorescence, Staining, Plasmid Preparation, Injection

Journal: Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/β-catenin/CCND1 signaling

doi: 10.1172/jci93198

Figure Lengend Snippet: Figure 5. LRP5 interacts with PDK1 to activate AKT signaling to inhibit GSK3β. (A) Protein levels of phospho-PDK1 (S241), PDK1, phospho-AKT (T308), AKT, phospho-GSK3β (S9), and GSK3β were measured in WT and Jam3-null YFP+Mac-1+c-Kit+ LICs by immunoblotting. (B) V5-tagged PDK1 and FLAG- tagged LRP5 were overexpressed in 293T cells, and their lysates were coimmunoprecipitated by V5 antibodies and protein A/G beads, followed by Western blotting analysis for FLAG (LRP5). (C) A reverse coimmunoprecipitation experiment was performed after LRP5-FLAG pull-down, followed by Western blotting analysis for PDK1 (V5). (D) A constitutively active form of phospho-AKT (E17K, AKTCN) was subcloned into pCDH-EF1a-T2A-mCherry vector and ectopically expressed in Jam3-null leukemia cells, followed by injection into recipient mice. Survival was compared among the mice receiving WT cells, Jam3-null cells, and AKTCN-overexpressing WT or Jam3-null cells (n = 5–7; **P < 0.01, ***P < 0.001, log-rank test). (E) Phospho-AKT (T308) and AKT levels were validated in leukemia cells from the rescue experiment in D. (F) The cell cycle distribution in YFP+Mac-1+c-Kit+ LICs from the rescue experiment in D was determined using Ki-67 and Hoechst 33342 staining (n = 3; ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). The empty vector was used as the control. Experiments were conducted 3 times for validation.

Article Snippet: Right after transplantation, 100 μg functional anti-JAM3 antibodies (catalog MCA5935XZ; clone CRAM-18 F26, Bio-Rad) (39, 40) or PBS were delivered into recipient mice via i.p. injection.

Techniques: Western Blot, Plasmid Preparation, Injection, Staining, Control, Biomarker Discovery

Journal: Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/β-catenin/CCND1 signaling

doi: 10.1172/jci93198

Figure Lengend Snippet: Figure 6. JAM3 is required for the proliferation of human leukemia cell lines. (A) Representative flow cytometric analysis of JAM3 expression on different leukemia cell lines including Kasumi-1 (M2), HL-60 (M3), THP-1 (M5), U937 (M5), and MV4-11 (M5). (Isotype control, gray line). (B) FLAG-tagged JAM3 and shRNAs targeting JAM3 (sh997, sh1188, sh359, and sh731) were cotransfected into 293T cells (1:4 ratio), followed by immunoblotting for JAM3. (C) Represen- tative images of JAM3-knockdown (sh731 and sh1188) THP-1 cells after 6 days in culture. (D–G) The numbers of THP-1, U937, Kasumi-1, and HL-60 cells were counted at the indicated days after infection with the JAM3-targeting sh731 or sh1188 or scrambled shRNA (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). (H) Representative images of colonies formed by the JAM3-knockdown (sh731 and sh1188) THP-1 cells after 9 days of culture in 1640 medium supplemented with 0.9% of methylcellulose and 10% of FBS. (I) Quantification of colony numbers in H (n = 3; **P < 0.01, ***P < 0.001, 1-way ANOVA followed by Bonferroni’s post-test). (J) Representative flow cytometric analysis of the cell cycle distribution in THP-1 cells target- ed by sh731, sh1188, or scrambled shRNA, which was determined using BrdU incorporation. (K) Quantitative analysis of the cell cycle distribution results in J (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). Experiments were conducted 3–5 times for validation.

Article Snippet: Right after transplantation, 100 μg functional anti-JAM3 antibodies (catalog MCA5935XZ; clone CRAM-18 F26, Bio-Rad) (39, 40) or PBS were delivered into recipient mice via i.p. injection.

Techniques: Expressing, Control, Western Blot, Knockdown, Infection, shRNA, BrdU Incorporation Assay, Biomarker Discovery

Journal: Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/β-catenin/CCND1 signaling

doi: 10.1172/jci93198

Figure Lengend Snippet: Figure 7. JAM3 supports the growth of human acute myeloid LICs. (A) Representative flow cytometric analysis of JAM3 expression on the immunopheno- typic Lin–CD34+CD38–CD90–CD45RA+ LICs (LMPP cells) and CD34–CD38– differentiated human AML cells (AML#7 in Supplemental Table 2). (B) Quantifica- tion of the MFIs for JAM3 expression on LMPP cells or CD34–CD38– differentiated leukemia cells in A (AML#2, #5, #6, #8 in Supplemental Table 2; n = 5; *P < 0.05, Student’s t test). (C) Quantification of the relative frequency of JAM3+ cells in LMPP or CD34–CD38– differentiated leukemia cells in A (n = 5; *P < 0.05, Student’s t test). (D–H) Cell numbers of 5 patient AML samples were counted at the indicated days after knockdown of JAM3 by sh1188 or scrambled shRNA (AML#1–AML#5 in Supplemental Table 2; n = 3; **P < 0.01, ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). Experi- ments were conducted 3–5 times for validation.

Article Snippet: Right after transplantation, 100 μg functional anti-JAM3 antibodies (catalog MCA5935XZ; clone CRAM-18 F26, Bio-Rad) (39, 40) or PBS were delivered into recipient mice via i.p. injection.

Techniques: Expressing, Knockdown, shRNA, Biomarker Discovery

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: The Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/ β -catenin/CCND1 signaling

doi: 10.1172/JCI93198

Figure Lengend Snippet: (A) mRNA levels of JAM3 in total BM cells, CMP, GMP, MPP, ST-HSCs, LT-HSCs, YFP+ leukemia cells, YFP+Mac-1+c-Kit+ LICs, and L-GMP cells was measured by quantitative RT-PCR (n = 3). (B–D) MLL-AF9+ leukemia cells were evaluated for LIC frequencies and c-Kit expression levels (MFI) in JAM3+ and JAM3– cells (n = 3; ***P < 0.001, Student’s t test). (E) Representative flow cytometric analysis of leukemia cells in the peripheral blood of recipient mice receiving transplants of WT or Jam3-null MLL-AF9+ BM cells upon the first to third transplantation. (F) Quantification data in E (n = 4–5; ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). PB, peripheral blood. (G–I) Survival data for recipient mice (lethally irradiated) receiving WT or Jam3-null MLL-AF9+ BM cells upon the first (G), second (H), and third transplantation (I) (n = 4–5; *P < 0.05, **P < 0.01, log-rank test). (J) Survival data for recipient mice (sublethally irradiated) receiving WT or Jam3-null leukemia cells upon the second transplantation (n = 5; ***P < 0.001, log-rank test). (K) Representative images of Giemsa-Wright staining for WT and Jam3-null MLL-AF9+ BM cells upon the second transplantation. (L) Quantification of the frequencies of blast cells in K (n = 3; ***P < 0.001, Student’s t test). (M) Representative images of the sizes of spleens and livers of recipient mice upon the second transplantation. (N and O) Quantification of the weight of spleens and livers in M (n = 4; *P < 0.05, **P < 0.01, Student’s t test). (P) Histological H&E staining of livers and spleens. (Q) Limiting dilution assays comparing the frequencies of LICs in WT and Jam3-null MLL-AF9+ BM cells. Experiments were conducted 3–5 times for validation.

Article Snippet: The expression levels of JAM3 in mouse or human LICs (or leukemia cell lines) were examined by anti–mouse JAM3–PE and anti–human JAM3–APC (eBioscience).

Techniques: Quantitative RT-PCR, Expressing, Transplantation Assay, Irradiation, Wright Stain, Staining

Journal: The Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/ β -catenin/CCND1 signaling

doi: 10.1172/JCI93198

Figure Lengend Snippet: (A) Representative flow cytometric analysis for WT and Jam3-null L-GMP cells of the recipients upon the secondary transplantation. (B) Quantification of frequencies of L-GMP cells in A (n = 3; ***P < 0.001, Student’s t test). (C and D) Survival data for recipient mice receiving WT or Jam3-null L-GMP cells upon the second to third transplantation (n = 5; **P < 0.01, log-rank test). (E–G) Representative images of colony formation of WT and Jam3-null YFP+Mac-1+c-Kit+ LICs of the secondary recipients in the first plating (E). The colony numbers (F) and total cell numbers of colonies in E (G) were counted (n = 3; ***P < 0.001, Student’s t test). (H–J) Representative images of colony formation of WT and Jam3-null leukemia cells clonogenically derived from the first plating (H). The colony numbers (I) and total cell numbers of colonies in H (J) were calculated (n = 3; ***P < 0.001, Student’s t test). (K) Cell cycle status was determined in WT and Jam3-null YFP+Mac-1+c-Kit+ LICs of the secondary recipients. (L) Quantitative analysis of the cell cycle distribution in K (n = 4–6; ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). (M) CFSE-labeled WT and Jam3-null leukemia cells of secondary recipients were transplanted and analyzed for the homed CFSE+ cells in the recipients’ BM and spleen (n = 5–6). (N) WT and Jam3-null leukemia cells of secondary recipients were transplanted into the recipient mice by intratibial injection, followed by the examination of leukemia cells 2 weeks later (n = 5; ***P < 0.001, Student’s t test). (O) Representative flow cytometric analysis of apoptosis of WT or Jam3-null YFP+Mac-1+c-Kit+ LICs. (P) Quantification of data in O (n = 4). Experiments were conducted 3–5 times for validation.

Article Snippet: The expression levels of JAM3 in mouse or human LICs (or leukemia cell lines) were examined by anti–mouse JAM3–PE and anti–human JAM3–APC (eBioscience).

Techniques: Transplantation Assay, Derivative Assay, Labeling, Injection

Journal: The Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/ β -catenin/CCND1 signaling

doi: 10.1172/JCI93198

Figure Lengend Snippet: (A and B) GO (biological process) and KEGG (pathway) analyses of the microarray data of WT or Jam3-null YFP+Mac-1+c-Kit+ LICs. Candidate changes are highlighted in red. (C) Potential candidates related to self-renewal, cell cycle, and Wnt signaling were examined in WT and Jam3-null LICs by quantitative RT-PCR (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test). (D) CCND1 levels were compared between WT and Jam3-null YFP+Mac-1+c-Kit+ LICs by immunoblotting. (E) Ccnd1 was ectopically expressed in Jam3-null leukemia cells and injected into recipient mice. Survival was compared among the mice receiving WT cells, Jam3-null cells, and Ccnd1-overexpressing WT or Jam3-null cells (n = 5–6; ***P < 0.001, log-rank test). (F) CCND1 levels were validated in leukemia cells from the rescue experiment in E. (G) The cell cycle distribution in YFP+Mac-1+c-Kit+ LICs from the rescue experiment in E was determined using Ki-67 and Hoechst 33342 staining (n = 3–5; *P < 0.05, **P < 0.01, 2-way ANOVA followed by Bonferroni’s post-test). Experiments were conducted 3–5 times for validation.

Article Snippet: The expression levels of JAM3 in mouse or human LICs (or leukemia cell lines) were examined by anti–mouse JAM3–PE and anti–human JAM3–APC (eBioscience).

Techniques: Microarray, Quantitative RT-PCR, Western Blot, Injection, Staining

Journal: The Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/ β -catenin/CCND1 signaling

doi: 10.1172/JCI93198

Figure Lengend Snippet: (A) Phospho–β-catenin (S552) and total β-catenin levels were evaluated between WT and Jam3-null YFP+Mac-1+c-Kit+ LICs by immunoblotting. (B) β-Catenin levels were compared between WT and Jam3-null YFP+Mac-1+c-Kit+ LICs by immunofluorescence staining. Scale bars: 5 µm. (C) A constitutively active form of phospho–β-catenin (S37A, β-cateninCN) was subcloned in the pCDH-EF1a-T2A-mCherry vector and ectopically expressed in Jam3-null leukemia cells, which were then injected into recipient mice. Survival was compared among the mice receiving WT cells, Jam3-null cells, and β-cateninCN–overexpressing WT or Jam3-null cells (n = 5–6; *P < 0.05, **P < 0.01, log-rank test). (D) Phospho–β-catenin (S552) and total β-catenin levels were validated in leukemia cells from the rescue experiment in C. (E) The cell cycle distribution in YFP+Mac-1+c-Kit+ LICs from the rescue experiment in C was determined using Ki-67 and Hoechst 33342 staining (n = 3; ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). (F) StrepII-tagged JAM3 and FLAG-tagged LRP5 were overexpressed in 293T cells, and their lysates were coimmunoprecipitated by strepII beads, followed by Western blotting analysis for FLAG (LRP5). (G) A reverse coimmunoprecipitation experiment was performed after LRP5-FLAG pull-down, followed by Western blotting analysis for strepII (JAM3). The empty vector was used as the control. Experiments were conducted 3 times for validation.

Article Snippet: The expression levels of JAM3 in mouse or human LICs (or leukemia cell lines) were examined by anti–mouse JAM3–PE and anti–human JAM3–APC (eBioscience).

Techniques: Western Blot, Immunofluorescence, Staining, Plasmid Preparation, Injection

Journal: The Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/ β -catenin/CCND1 signaling

doi: 10.1172/JCI93198

Figure Lengend Snippet: (A) Protein levels of phospho-PDK1 (S241), PDK1, phospho-AKT (T308), AKT, phospho-GSK3β (S9), and GSK3β were measured in WT and Jam3-null YFP+Mac-1+c-Kit+ LICs by immunoblotting. (B) V5-tagged PDK1 and FLAG-tagged LRP5 were overexpressed in 293T cells, and their lysates were coimmunoprecipitated by V5 antibodies and protein A/G beads, followed by Western blotting analysis for FLAG (LRP5). (C) A reverse coimmunoprecipitation experiment was performed after LRP5-FLAG pull-down, followed by Western blotting analysis for PDK1 (V5). (D) A constitutively active form of phospho-AKT (E17K, AKTCN) was subcloned into pCDH-EF1a-T2A-mCherry vector and ectopically expressed in Jam3-null leukemia cells, followed by injection into recipient mice. Survival was compared among the mice receiving WT cells, Jam3-null cells, and AKTCN-overexpressing WT or Jam3-null cells (n = 5–7; **P < 0.01, ***P < 0.001, log-rank test). (E) Phospho-AKT (T308) and AKT levels were validated in leukemia cells from the rescue experiment in D. (F) The cell cycle distribution in YFP+Mac-1+c-Kit+ LICs from the rescue experiment in D was determined using Ki-67 and Hoechst 33342 staining (n = 3; ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). The empty vector was used as the control. Experiments were conducted 3 times for validation.

Article Snippet: The expression levels of JAM3 in mouse or human LICs (or leukemia cell lines) were examined by anti–mouse JAM3–PE and anti–human JAM3–APC (eBioscience).

Techniques: Western Blot, Plasmid Preparation, Injection, Staining

Journal: The Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/ β -catenin/CCND1 signaling

doi: 10.1172/JCI93198

Figure Lengend Snippet: (A) Representative flow cytometric analysis of JAM3 expression on different leukemia cell lines including Kasumi-1 (M2), HL-60 (M3), THP-1 (M5), U937 (M5), and MV4-11 (M5). (Isotype control, gray line). (B) FLAG-tagged JAM3 and shRNAs targeting JAM3 (sh997, sh1188, sh359, and sh731) were cotransfected into 293T cells (1:4 ratio), followed by immunoblotting for JAM3. (C) Representative images of JAM3-knockdown (sh731 and sh1188) THP-1 cells after 6 days in culture. (D–G) The numbers of THP-1, U937, Kasumi-1, and HL-60 cells were counted at the indicated days after infection with the JAM3-targeting sh731 or sh1188 or scrambled shRNA (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). (H) Representative images of colonies formed by the JAM3-knockdown (sh731 and sh1188) THP-1 cells after 9 days of culture in 1640 medium supplemented with 0.9% of methylcellulose and 10% of FBS. (I) Quantification of colony numbers in H (n = 3; **P < 0.01, ***P < 0.001, 1-way ANOVA followed by Bonferroni’s post-test). (J) Representative flow cytometric analysis of the cell cycle distribution in THP-1 cells targeted by sh731, sh1188, or scrambled shRNA, which was determined using BrdU incorporation. (K) Quantitative analysis of the cell cycle distribution results in J (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). Experiments were conducted 3–5 times for validation.

Article Snippet: The expression levels of JAM3 in mouse or human LICs (or leukemia cell lines) were examined by anti–mouse JAM3–PE and anti–human JAM3–APC (eBioscience).

Techniques: Expressing, Western Blot, Infection, shRNA, BrdU Incorporation Assay

Journal: The Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/ β -catenin/CCND1 signaling

doi: 10.1172/JCI93198

Figure Lengend Snippet: (A) Representative flow cytometric analysis of JAM3 expression on the immunophenotypic Lin–CD34+CD38–CD90–CD45RA+ LICs (LMPP cells) and CD34–CD38– differentiated human AML cells (AML#7 in Supplemental Table 2). (B) Quantification of the MFIs for JAM3 expression on LMPP cells or CD34–CD38– differentiated leukemia cells in A (AML#2, #5, #6, #8 in Supplemental Table 2; n = 5; *P < 0.05, Student’s t test). (C) Quantification of the relative frequency of JAM3+ cells in LMPP or CD34–CD38– differentiated leukemia cells in A (n = 5; *P < 0.05, Student’s t test). (D–H) Cell numbers of 5 patient AML samples were counted at the indicated days after knockdown of JAM3 by sh1188 or scrambled shRNA (AML#1–AML#5 in Supplemental Table 2; n = 3; **P < 0.01, ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). Experiments were conducted 3–5 times for validation.

Article Snippet: The expression levels of JAM3 in mouse or human LICs (or leukemia cell lines) were examined by anti–mouse JAM3–PE and anti–human JAM3–APC (eBioscience).

Techniques: Expressing, shRNA

Journal: The Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/ β -catenin/CCND1 signaling

doi: 10.1172/JCI93198

Figure Lengend Snippet: (A) mRNA levels of JAM3 in total BM cells, CMP, GMP, MPP, ST-HSCs, LT-HSCs, YFP+ leukemia cells, YFP+Mac-1+c-Kit+ LICs, and L-GMP cells was measured by quantitative RT-PCR (n = 3). (B–D) MLL-AF9+ leukemia cells were evaluated for LIC frequencies and c-Kit expression levels (MFI) in JAM3+ and JAM3– cells (n = 3; ***P < 0.001, Student’s t test). (E) Representative flow cytometric analysis of leukemia cells in the peripheral blood of recipient mice receiving transplants of WT or Jam3-null MLL-AF9+ BM cells upon the first to third transplantation. (F) Quantification data in E (n = 4–5; ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). PB, peripheral blood. (G–I) Survival data for recipient mice (lethally irradiated) receiving WT or Jam3-null MLL-AF9+ BM cells upon the first (G), second (H), and third transplantation (I) (n = 4–5; *P < 0.05, **P < 0.01, log-rank test). (J) Survival data for recipient mice (sublethally irradiated) receiving WT or Jam3-null leukemia cells upon the second transplantation (n = 5; ***P < 0.001, log-rank test). (K) Representative images of Giemsa-Wright staining for WT and Jam3-null MLL-AF9+ BM cells upon the second transplantation. (L) Quantification of the frequencies of blast cells in K (n = 3; ***P < 0.001, Student’s t test). (M) Representative images of the sizes of spleens and livers of recipient mice upon the second transplantation. (N and O) Quantification of the weight of spleens and livers in M (n = 4; *P < 0.05, **P < 0.01, Student’s t test). (P) Histological H&E staining of livers and spleens. (Q) Limiting dilution assays comparing the frequencies of LICs in WT and Jam3-null MLL-AF9+ BM cells. Experiments were conducted 3–5 times for validation.

Article Snippet: The expression levels of JAM3 in mouse or human LICs (or leukemia cell lines) were examined by anti–mouse JAM3–PE and anti–human JAM3–APC (eBioscience).

Techniques: Quantitative RT-PCR, Expressing, Transplantation Assay, Irradiation, Wright Stain, Staining

Journal: The Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/ β -catenin/CCND1 signaling

doi: 10.1172/JCI93198

Figure Lengend Snippet: (A) Representative flow cytometric analysis for WT and Jam3-null L-GMP cells of the recipients upon the secondary transplantation. (B) Quantification of frequencies of L-GMP cells in A (n = 3; ***P < 0.001, Student’s t test). (C and D) Survival data for recipient mice receiving WT or Jam3-null L-GMP cells upon the second to third transplantation (n = 5; **P < 0.01, log-rank test). (E–G) Representative images of colony formation of WT and Jam3-null YFP+Mac-1+c-Kit+ LICs of the secondary recipients in the first plating (E). The colony numbers (F) and total cell numbers of colonies in E (G) were counted (n = 3; ***P < 0.001, Student’s t test). (H–J) Representative images of colony formation of WT and Jam3-null leukemia cells clonogenically derived from the first plating (H). The colony numbers (I) and total cell numbers of colonies in H (J) were calculated (n = 3; ***P < 0.001, Student’s t test). (K) Cell cycle status was determined in WT and Jam3-null YFP+Mac-1+c-Kit+ LICs of the secondary recipients. (L) Quantitative analysis of the cell cycle distribution in K (n = 4–6; ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). (M) CFSE-labeled WT and Jam3-null leukemia cells of secondary recipients were transplanted and analyzed for the homed CFSE+ cells in the recipients’ BM and spleen (n = 5–6). (N) WT and Jam3-null leukemia cells of secondary recipients were transplanted into the recipient mice by intratibial injection, followed by the examination of leukemia cells 2 weeks later (n = 5; ***P < 0.001, Student’s t test). (O) Representative flow cytometric analysis of apoptosis of WT or Jam3-null YFP+Mac-1+c-Kit+ LICs. (P) Quantification of data in O (n = 4). Experiments were conducted 3–5 times for validation.

Article Snippet: The expression levels of JAM3 in mouse or human LICs (or leukemia cell lines) were examined by anti–mouse JAM3–PE and anti–human JAM3–APC (eBioscience).

Techniques: Transplantation Assay, Derivative Assay, Labeling, Injection

Journal: The Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/ β -catenin/CCND1 signaling

doi: 10.1172/JCI93198

Figure Lengend Snippet: (A and B) GO (biological process) and KEGG (pathway) analyses of the microarray data of WT or Jam3-null YFP+Mac-1+c-Kit+ LICs. Candidate changes are highlighted in red. (C) Potential candidates related to self-renewal, cell cycle, and Wnt signaling were examined in WT and Jam3-null LICs by quantitative RT-PCR (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test). (D) CCND1 levels were compared between WT and Jam3-null YFP+Mac-1+c-Kit+ LICs by immunoblotting. (E) Ccnd1 was ectopically expressed in Jam3-null leukemia cells and injected into recipient mice. Survival was compared among the mice receiving WT cells, Jam3-null cells, and Ccnd1-overexpressing WT or Jam3-null cells (n = 5–6; ***P < 0.001, log-rank test). (F) CCND1 levels were validated in leukemia cells from the rescue experiment in E. (G) The cell cycle distribution in YFP+Mac-1+c-Kit+ LICs from the rescue experiment in E was determined using Ki-67 and Hoechst 33342 staining (n = 3–5; *P < 0.05, **P < 0.01, 2-way ANOVA followed by Bonferroni’s post-test). Experiments were conducted 3–5 times for validation.

Article Snippet: The expression levels of JAM3 in mouse or human LICs (or leukemia cell lines) were examined by anti–mouse JAM3–PE and anti–human JAM3–APC (eBioscience).

Techniques: Microarray, Quantitative RT-PCR, Western Blot, Injection, Staining

Journal: The Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/ β -catenin/CCND1 signaling

doi: 10.1172/JCI93198

Figure Lengend Snippet: (A) Phospho–β-catenin (S552) and total β-catenin levels were evaluated between WT and Jam3-null YFP+Mac-1+c-Kit+ LICs by immunoblotting. (B) β-Catenin levels were compared between WT and Jam3-null YFP+Mac-1+c-Kit+ LICs by immunofluorescence staining. Scale bars: 5 µm. (C) A constitutively active form of phospho–β-catenin (S37A, β-cateninCN) was subcloned in the pCDH-EF1a-T2A-mCherry vector and ectopically expressed in Jam3-null leukemia cells, which were then injected into recipient mice. Survival was compared among the mice receiving WT cells, Jam3-null cells, and β-cateninCN–overexpressing WT or Jam3-null cells (n = 5–6; *P < 0.05, **P < 0.01, log-rank test). (D) Phospho–β-catenin (S552) and total β-catenin levels were validated in leukemia cells from the rescue experiment in C. (E) The cell cycle distribution in YFP+Mac-1+c-Kit+ LICs from the rescue experiment in C was determined using Ki-67 and Hoechst 33342 staining (n = 3; ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). (F) StrepII-tagged JAM3 and FLAG-tagged LRP5 were overexpressed in 293T cells, and their lysates were coimmunoprecipitated by strepII beads, followed by Western blotting analysis for FLAG (LRP5). (G) A reverse coimmunoprecipitation experiment was performed after LRP5-FLAG pull-down, followed by Western blotting analysis for strepII (JAM3). The empty vector was used as the control. Experiments were conducted 3 times for validation.

Article Snippet: The expression levels of JAM3 in mouse or human LICs (or leukemia cell lines) were examined by anti–mouse JAM3–PE and anti–human JAM3–APC (eBioscience).

Techniques: Western Blot, Immunofluorescence, Staining, Plasmid Preparation, Injection

Journal: The Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/ β -catenin/CCND1 signaling

doi: 10.1172/JCI93198

Figure Lengend Snippet: (A) Protein levels of phospho-PDK1 (S241), PDK1, phospho-AKT (T308), AKT, phospho-GSK3β (S9), and GSK3β were measured in WT and Jam3-null YFP+Mac-1+c-Kit+ LICs by immunoblotting. (B) V5-tagged PDK1 and FLAG-tagged LRP5 were overexpressed in 293T cells, and their lysates were coimmunoprecipitated by V5 antibodies and protein A/G beads, followed by Western blotting analysis for FLAG (LRP5). (C) A reverse coimmunoprecipitation experiment was performed after LRP5-FLAG pull-down, followed by Western blotting analysis for PDK1 (V5). (D) A constitutively active form of phospho-AKT (E17K, AKTCN) was subcloned into pCDH-EF1a-T2A-mCherry vector and ectopically expressed in Jam3-null leukemia cells, followed by injection into recipient mice. Survival was compared among the mice receiving WT cells, Jam3-null cells, and AKTCN-overexpressing WT or Jam3-null cells (n = 5–7; **P < 0.01, ***P < 0.001, log-rank test). (E) Phospho-AKT (T308) and AKT levels were validated in leukemia cells from the rescue experiment in D. (F) The cell cycle distribution in YFP+Mac-1+c-Kit+ LICs from the rescue experiment in D was determined using Ki-67 and Hoechst 33342 staining (n = 3; ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). The empty vector was used as the control. Experiments were conducted 3 times for validation.

Article Snippet: The expression levels of JAM3 in mouse or human LICs (or leukemia cell lines) were examined by anti–mouse JAM3–PE and anti–human JAM3–APC (eBioscience).

Techniques: Western Blot, Plasmid Preparation, Injection, Staining

Journal: The Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/ β -catenin/CCND1 signaling

doi: 10.1172/JCI93198

Figure Lengend Snippet: (A) Representative flow cytometric analysis of JAM3 expression on different leukemia cell lines including Kasumi-1 (M2), HL-60 (M3), THP-1 (M5), U937 (M5), and MV4-11 (M5). (Isotype control, gray line). (B) FLAG-tagged JAM3 and shRNAs targeting JAM3 (sh997, sh1188, sh359, and sh731) were cotransfected into 293T cells (1:4 ratio), followed by immunoblotting for JAM3. (C) Representative images of JAM3-knockdown (sh731 and sh1188) THP-1 cells after 6 days in culture. (D–G) The numbers of THP-1, U937, Kasumi-1, and HL-60 cells were counted at the indicated days after infection with the JAM3-targeting sh731 or sh1188 or scrambled shRNA (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). (H) Representative images of colonies formed by the JAM3-knockdown (sh731 and sh1188) THP-1 cells after 9 days of culture in 1640 medium supplemented with 0.9% of methylcellulose and 10% of FBS. (I) Quantification of colony numbers in H (n = 3; **P < 0.01, ***P < 0.001, 1-way ANOVA followed by Bonferroni’s post-test). (J) Representative flow cytometric analysis of the cell cycle distribution in THP-1 cells targeted by sh731, sh1188, or scrambled shRNA, which was determined using BrdU incorporation. (K) Quantitative analysis of the cell cycle distribution results in J (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). Experiments were conducted 3–5 times for validation.

Article Snippet: The expression levels of JAM3 in mouse or human LICs (or leukemia cell lines) were examined by anti–mouse JAM3–PE and anti–human JAM3–APC (eBioscience).

Techniques: Expressing, Western Blot, Infection, shRNA, BrdU Incorporation Assay

Journal: The Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/ β -catenin/CCND1 signaling

doi: 10.1172/JCI93198

Figure Lengend Snippet: (A) Representative flow cytometric analysis of JAM3 expression on the immunophenotypic Lin–CD34+CD38–CD90–CD45RA+ LICs (LMPP cells) and CD34–CD38– differentiated human AML cells (AML#7 in Supplemental Table 2). (B) Quantification of the MFIs for JAM3 expression on LMPP cells or CD34–CD38– differentiated leukemia cells in A (AML#2, #5, #6, #8 in Supplemental Table 2; n = 5; *P < 0.05, Student’s t test). (C) Quantification of the relative frequency of JAM3+ cells in LMPP or CD34–CD38– differentiated leukemia cells in A (n = 5; *P < 0.05, Student’s t test). (D–H) Cell numbers of 5 patient AML samples were counted at the indicated days after knockdown of JAM3 by sh1188 or scrambled shRNA (AML#1–AML#5 in Supplemental Table 2; n = 3; **P < 0.01, ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). Experiments were conducted 3–5 times for validation.

Article Snippet: The expression levels of JAM3 in mouse or human LICs (or leukemia cell lines) were examined by anti–mouse JAM3–PE and anti–human JAM3–APC (eBioscience).

Techniques: Expressing, shRNA