Journal: Frontiers in Cell and Developmental Biology

Article Title: HAX1 , gene responsible for Kostmann syndrome, regulates gingival epithelial barrier function via intracellular trafficking of JAM1

doi: 10.3389/fcell.2025.1624718

Figure Lengend Snippet: HAX1 knockdown decreases JAM1 expression in IHGE cells. (A) RT-PCR analysis of HAX1 gene in IHGE cells. Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) was used as the control. (B) The level of HAX1 mRNA expression in IHGE cells transfected with siControl or siHAX1 (#1, #2) are expressed as fold change relative to siControl-transfected cells, with mean results (bars) of five technical replicates shown. (C) IHGE cells transfected with siControl or siHAX1 (#1, #2) were fixed, then stained with DAPI (cyan) and anti-TOMM20, and analyzed by confocal microscopy. Scale bars, 10 μm. (D) IHGE cells transfected with siControl or siHAX1 (#1, #2) were analyzed by immunoblotting with the indicated antibodies. IB, immunoblot. (E) IHGE cells transfected with siControl or siHAX1 (#1, #2) were fixed, then stained with DAPI (cyan) and anti-JAM1 (yellow: Alexa Fluor 555), and analyzed by confocal microscopy. Scale bars, 10 μm. See also .

Article Snippet: HAX1 KO IHGE cells stably expressing JAM1 were selected with use of G418 (ant-gen-1, 200 μg mL −1 ) (InvivoGen).

Techniques: Knockdown, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Transfection, Staining, Confocal Microscopy, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: HAX1 , gene responsible for Kostmann syndrome, regulates gingival epithelial barrier function via intracellular trafficking of JAM1

doi: 10.3389/fcell.2025.1624718

Figure Lengend Snippet: Cisplatin decreases HAX1 and JAM1 expression in IHGE cells. (A) IHGE cells treated with or without cisplatin (20 μM) for 24 h were analyzed by immunoblotting with the indicated antibodies. (B,C) IHGE cells treated with or without cisplatin (20 μM) for 24 h were fixed, then stained with DAPI (cyan) and either anti-TOMM20 [gray: Alexa Fluor 555 in (B) ] or anti-JAM1 [gray: Alexa Fluor 555 in (C) ], and analyzed by confocal microscopy. Scale bars, 10 μm.

Article Snippet: HAX1 KO IHGE cells stably expressing JAM1 were selected with use of G418 (ant-gen-1, 200 μg mL −1 ) (InvivoGen).

Techniques: Expressing, Western Blot, Staining, Confocal Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: HAX1 , gene responsible for Kostmann syndrome, regulates gingival epithelial barrier function via intracellular trafficking of JAM1

doi: 10.3389/fcell.2025.1624718

Figure Lengend Snippet: Effects of HAX1 Q190X mutation on JAM1 expression in IHGE cells. (A) IHGE WT cells and with HAX1 KO were separately analyzed by immunoblotting with the indicated antibodies. (B) IHGE cells were transfected with plasmid coding EGFP-HAX1 WT or Q190X (green). At 3 days after transfection, the cells were fixed, stained with anti-TOMM20 (magenta: Alexa Fluor 555), and analyzed by confocal microscopy. Scale bars, 10 μm. (C) IHGE HAX1 KO cells were transfected with plasmid coding EGFP-HAX1 WT or HAX1 Q190X. At 3 days after transfection, the cells were analyzed by immunoblotting with the indicated antibodies.

Article Snippet: HAX1 KO IHGE cells stably expressing JAM1 were selected with use of G418 (ant-gen-1, 200 μg mL −1 ) (InvivoGen).

Techniques: Mutagenesis, Expressing, Western Blot, Transfection, Plasmid Preparation, Staining, Confocal Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: HAX1 , gene responsible for Kostmann syndrome, regulates gingival epithelial barrier function via intracellular trafficking of JAM1

doi: 10.3389/fcell.2025.1624718

Figure Lengend Snippet: Effects of bafilomycin A1 on JAM1 in IHGE HAX1 KO cells. (A,B) JAM1 mRNA expressions in IHGE WT cells and with HAX1 KO are expressed as fold change relative to WT cells, with mean results (bars) of three technical replicates shown (A) . JAM1 mRNA expressions in IHGE HAX1 KO cells transfected with a plasmid coding EGFP-tagged HAX1 WT or Q190X, or an empty vector are expressed as fold change relative to control cells (empty vector), with mean results (bars) of three technical replicates shown (B) . β-ACTIN was used as the control. β-ACTIN was used as the control. Results are expressed as fold change relative to WT cells and presented as the mean ± SD of three technical replicates. *p < 0.05, two-tailed t -test. (C) IHGE HAX1 KO cells were treated with bafilomycin A1 (5 nM). At 12 h after administration, the cells were fixed, then stained with DAPI (cyan) and anti-JAM1 (gray: Alexa Fluor 555), and analyzed by confocal microscopy. Scale bars, 10 μm. (D) IHGE HAX1 KO cells were treated with bafilomycin A1 (10 nM). At 24 h after administration, the cells were stained with anti-JAM1 (gray, Alexa Fluor 555) and anti-LAMP1 (magenta, Alexa Fluor 647) and, then analyzed by confocal microscopy. Magnified image (lower panel) was captured in a zoomed condition with the microscopic software in the different area from the upper panel. Scale bars, 10 μm.

Article Snippet: HAX1 KO IHGE cells stably expressing JAM1 were selected with use of G418 (ant-gen-1, 200 μg mL −1 ) (InvivoGen).

Techniques: Transfection, Plasmid Preparation, Control, Two Tailed Test, Staining, Confocal Microscopy, Software

Journal: Frontiers in Cell and Developmental Biology

Article Title: HAX1 , gene responsible for Kostmann syndrome, regulates gingival epithelial barrier function via intracellular trafficking of JAM1

doi: 10.3389/fcell.2025.1624718

Figure Lengend Snippet: JAM1 involved in disruption of barrier against LPS and PGN caused by HAX1 KO in IHGE cells. (A) Schematic image of culture-insert system. Monolayers of IHGE WT and HAX1 KO cells with or without overexpression of JAM1 were separately cultured in the upper compartments. Fluorescent tracers were added and culturing was performed for 30 min, after which culture medium was obtained from the lower compartment and analyzed using spectrometry. (B) Representative confocal microscopic images of IHGE WT and HAX1 KO cells with or without overexpression of JAM1 (HA-inserted JAM1). Alexa Fluor 633-conjugated phalloidin (cyan) and anti-JAM1 (magenta: Alexa Fluor 555) staining was performed. Scale bars, 10 μm. See also . (C–E) IHGE cell permeability to FITC-40 kDa dextran (C) , P. gingivalis LPS (D) , and FITC- S. aureus PGN (E) . Results are expressed as fold change relative to WT cells and presented as the mean ± SD of eight technical replicates. *p < 0.05, two-tailed t -test (closed testing procedure). OE, overexpression.

Article Snippet: HAX1 KO IHGE cells stably expressing JAM1 were selected with use of G418 (ant-gen-1, 200 μg mL −1 ) (InvivoGen).

Techniques: Disruption, Over Expression, Cell Culture, Staining, Permeability, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: HAX1 , gene responsible for Kostmann syndrome, regulates gingival epithelial barrier function via intracellular trafficking of JAM1

doi: 10.3389/fcell.2025.1624718

Figure Lengend Snippet: HAX1 KO reduces barrier function of gingival epithelial tissues. (A) Schematic image of culture-insert system. WT and HAX1 KO gingival epithelial tissues with or without overexpression of JAM1 were separately cultured in the upper compartments. FITC-labeled tracers were then added to medium in each upper compartment. Following 6 h of incubation, tracer movement from the upper to lower compartment was analyzed by spectrometry. (B) Confocal microscopic cross-sectional images of 3D-tissue models of IHGE WT cells and those with HAX1 KO. Gingival epithelial WT and HAX1 KO tissues with or without overexpression of JAM1 on coverslips were separately fixed, then stained with DAPI (cyan) and Alexa Fluor 633-conjugated phalloidin (gray), and analyzed using confocal microscopy. Scale bars, 30 μm. (C,D) Permeability of gingival epithelial tissues to FITC- P. gingivalis LPS (C) or S. aureus PGN (D) . Results expressed as fold change relative to the control (WT tissues) were obtained and are presented as the mean ± SD of eight technical replicates. *p < 0.05, two-tailed t -test (closed testing procedure).

Article Snippet: HAX1 KO IHGE cells stably expressing JAM1 were selected with use of G418 (ant-gen-1, 200 μg mL −1 ) (InvivoGen).

Techniques: Over Expression, Cell Culture, Labeling, Incubation, Staining, Confocal Microscopy, Permeability, Control, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: HAX1 , gene responsible for Kostmann syndrome, regulates gingival epithelial barrier function via intracellular trafficking of JAM1

doi: 10.3389/fcell.2025.1624718

Figure Lengend Snippet: Cisplatin dampens epithelial barrier function of gingival epithelial tissues. (A) Schematic image of culture-insert system. Cisplatin-treated gingival epithelial tissues with or without overexpression of JAM1 were cultured in the upper compartments. At 24 h after administration, FITC-labeled tracers were added to culture medium in each upper compartment. Following 6 h of incubation, tracer movement from the upper to lower compartment was analyzed using spectrometry. (B) Confocal microscopic cross-sectional images of 3D-tissue model of IHGE cells. Gingival epithelial tissues with or without overexpression of JAM1 were treated with cisplatin (20 μM). At 24 h after administration, cells on coverslips were fixed, then stained with DAPI (cyan) and Alexa Fluor 633-conjugated phalloidin (gray), and analyzed using confocal microscopy. Scale bars, 30 μm. (C,D) Permeability of gingival epithelial tissues to FITC- P. gingivalis LPS (C) or S. aureus PGN (D) . Results expressed as fold change relative to the control (WT tissues without cisplatin) were obtained and are presented as the mean ± SD of eight technical replicates. *p < 0.05, two-tailed t -test (closed-testing procedure).

Article Snippet: HAX1 KO IHGE cells stably expressing JAM1 were selected with use of G418 (ant-gen-1, 200 μg mL −1 ) (InvivoGen).

Techniques: Over Expression, Cell Culture, Labeling, Incubation, Staining, Confocal Microscopy, Permeability, Control, Two Tailed Test

Journal: Cell Regeneration

Article Title: Mammalian mesenchymal stromal cells enhance zebrafish fin regeneration

doi: 10.1186/s13619-025-00273-7

Figure Lengend Snippet: Murine MSCs treated with indomethacin are more susceptible to phagocytosis by activated primary macrophages. A Experimental design: bone marrow-derived monocytes were differentiated into macrophages (Mφ) with M-CSF. Macrophages were then activated with LPS + IFN-γ to induce a pro-inflammatory M1-like phenotype. Activated macrophages were co-cultured in direct contact with control or indomethacin-treated MSCs. Macrophage phenotype was analyzed by flow cytometry (FACS), with all cells gated on the CD45 + CD11b + F4/80 + population, representing differentiated macrophages. B - C Median fluorescence intensity (MFI) of DiD, indicating phagocytosis, was measured in the pro-inflammatory macrophage subpopulations MHC-II + CD86 + ( B ) and MHC-II + CD80 + macrophages ( C ). Representative histograms are shown alongside MFI values. Data are shown as mean ± SD. n = 3 independent experiments. Statistical significance was assessed using one-way ANOVA. *: p < 0.05; **: p < 0.005; ***: p < 0.001; ****: p < 0.0001

Article Snippet: Subsequently, cells were stained with specific antibodies targeting CD45 (clone 30-F11), CD11b (clone M1/70), CD80 (clone 16-10A1) (BD Biosciences), MHC-II (clone M5/114.15.2) (Miltenyi Biotec, Germany), and F4/80 (clone BM8) (eBioscience, USA).

Techniques: Derivative Assay, Cell Culture, Control, Flow Cytometry, Fluorescence