Journal: Journal of Cellular and Molecular Medicine

Article Title: Long non‐coding RNA‐H19 stimulates osteogenic differentiation of bone marrow mesenchymal stem cells via the microRNA‐149/ SDF‐1 axis

doi: 10.1111/jcmm.15040

Figure Lengend Snippet: BMMSCs of rats are successfully isolated and cultured. A, Morphology of BMMSCs at P0, P1 and P3 observed under an inverted fluorescence microscopy (200×). B, Flow cytometry detection of the surface markers of BMMSCs (CD29, CD44, CD90 and CD45). C, adipogenic differentiation of BMMSCs was visualized by Oil red O staining (400×). D, Osteogenic differentiation of BMMSCs was visualized by alizarin red S (200×)

Article Snippet: Cell suspensions of a density of 1 × 10 6 cells/L were incubated with antibodies against CD29 phycoerythrin (PE; 562154, 1:100, 20 µL, BD Biosciences), CD44‐PE (ab23396, 1:200, 20 µL, Abcam), CD90‐PE (551401, 1:100, 20 µL, BD Biosciences) and CD45‐PE (554878, 1:100, 20 µL, BD Biosciences).

Techniques: Isolation, Cell Culture, Fluorescence, Microscopy, Flow Cytometry, Staining

Journal: Journal of Translational Medicine

Article Title: Enhanced cytotoxicity against cholangiocarcinoma by fifth-generation chimeric antigen receptor T cells targeting integrin αvβ6 and secreting anti-PD-L1 scFv

doi: 10.1186/s12967-025-06453-y

Figure Lengend Snippet: A20 CAR5 T cells infiltrate integrin αvβ6 + /PD-L1 + KKU-213A spheroids. A Representative images were captured to visualize the infiltration of A20 CAR T cells into the spheroids. In brief, 4 × 10 3 cells of KKU-213A labeled with CellTracker ™ Blue 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (CMF 2 HC) were co-cultured with either NT T, A20 CAR4 T or A20 CAR5 T cells labeled with CFSE (Thermo Fisher Scientific) at an E:T ratio of 2:1. B Histograms represent the averaged intensity profiles of blue CMF 2 HC KKU-213A spheroids and green CFSE T cells signals in z-stack cross-sections of spheroids defied by ImageJ. C T cell infiltration was quantified by calculating the area under the curve (AUC) of the green CFSE signals. Scale bar = 200 μm. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test (** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: The cells were then incubated overnight at 4 °C with mouse anti-integrin β6 antibody (1:100 dilution, Clone 442.5C4; Merck Millipore, Massachusetts, USA) and rabbit anti-PD-L1 antibody (1:100 dilution, Clone 28–8, Abcam, Cambridge, UK).

Techniques: Labeling, Cell Culture

Journal: Journal of Translational Medicine

Article Title: Enhanced cytotoxicity against cholangiocarcinoma by fifth-generation chimeric antigen receptor T cells targeting integrin αvβ6 and secreting anti-PD-L1 scFv

doi: 10.1186/s12967-025-06453-y

Figure Lengend Snippet: Expression of integrin αvβ6 and PD-L1 in cholangiocarcinoma (CCA) cell lines. A Immunofluorescence analysis showing the expression of integrin αvβ6 (green) and PD-L1 (red) in three CCA cell lines: KKU-055, KKU-100, and KKU-213A. The scale bar represents 50 μm. B Immunoblot analysis detecting PD-L1 ( ~ 55 kDa) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 37 kDa) as a loading control (upper panel). Densitometry quantification of PD-L1 relative to GAPDH was performed using ImageJ software (lower panel). C Flow cytometry histograms displaying the surface expression of integrin αvβ6 and ( E ) PD-L1, with isotype controls shown in light gray. Quantification of ( D ) integrin αvβ6- and ( F ) PD-L1-positive cells is presented as percentages. Data are shown as mean ± standard error of the mean (SEM) from three independent experiments (N = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test (* p < 0.05, *** p < 0.001, **** p < 0.0001)

Article Snippet: The cells were then incubated overnight at 4 °C with mouse anti-integrin β6 antibody (1:100 dilution, Clone 442.5C4; Merck Millipore, Massachusetts, USA) and rabbit anti-PD-L1 antibody (1:100 dilution, Clone 28–8, Abcam, Cambridge, UK).

Techniques: Expressing, Immunofluorescence, Western Blot, Control, Software, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: Enhanced cytotoxicity against cholangiocarcinoma by fifth-generation chimeric antigen receptor T cells targeting integrin αvβ6 and secreting anti-PD-L1 scFv

doi: 10.1186/s12967-025-06453-y

Figure Lengend Snippet: Schematic illustrating the mechanism of A20 CAR5 T cells, designed to mimic the natural activation process of T cells. The integrin αvβ6-targeted CAR recognizes and binds to integrin αvβ6 present on cancer cells, while PD-L1, an immune checkpoint molecule, inhibits T cell activity by interacting with PD-1 expressed on the T cell surface. Full T cell activation occurs when integrin αvβ6 is recognized, and PD-L1 is neutralized by the secreted anti-PD-L1 scFv. Without this neutralization, PD-L1 remains active, preventing full T cell activation and reducing the immune response

Article Snippet: The cells were then incubated overnight at 4 °C with mouse anti-integrin β6 antibody (1:100 dilution, Clone 442.5C4; Merck Millipore, Massachusetts, USA) and rabbit anti-PD-L1 antibody (1:100 dilution, Clone 28–8, Abcam, Cambridge, UK).

Techniques: Activation Assay, Activity Assay, Neutralization

Journal: Developmental Cell

Article Title: Integrin-Mediated Focal Anchorage Drives Epithelial Zippering during Mouse Neural Tube Closure

doi: 10.1016/j.devcel.2020.01.012

Figure Lengend Snippet: Integrin Expression and Localization during Spinal NT Closure (A and B) RNA-seq analysis of integrin subunit expression in the caudal region of E9.5 (20 som) stage embryos (A). Schematic of integrin subunits expressed and functional interactions (B). Integrin β1 is the most highly expressed subunit and forms integrins α5β1 and αvβ1, which bind fibronectin (green), and integrins α3β1 and α6β1, which bind laminin (red). Subunit β5 pairs exclusively with αv to mediate binding to vitronectin, which is not expressed at this stage. The receptor α9β1 interacts with vitronectin and tenascin-C but neither are expressed. No collagen IV-interacting subunits are expressed. (C) In situ hybridization analysis of integrin subunit gene expression. Itgβ1 and Itgα5 show an intense signal at the site of zippering where the neural fold tips come into contact (insets). Itgαv and Itgα6 both show a ventro-dorsal gradient of expression in the NE, with Itgα6 expression also in the SE. Itgα3 is expressed in the SE only. Stages: β1 (19 som), αv (19 som), α5 (20 som), α3 (16 som), and α6 (19 som). (D) Immunofluorescence showing expression of Itgβ1 and Itgα5 specifically at the zippering point (arrows). (E) Diagram of neural fold tips indicating neuroepithelial (top) and SE (bottom) potential interactions of integrin α5β1 with the intervening fibronectin BM. Scale bars: 100 μm (C); 100 μm (D); and 50 μm (D) (zoom). See also Figure S2 .

Article Snippet: Antibodies : Two different antibodies were used to detect the integrin β1 receptor: a rat monoclonal anti-Integrin β1 (MAB1997) that recognises the full β1 subunit and a rat monoclonal anti-Integrin β1 (BD Biosciences, 553715) that has been reported to recognise the active/ligand-bound form of the β1 subunit ( , ).

Techniques: Expressing, RNA Sequencing Assay, Functional Assay, Binding Assay, In Situ Hybridization, Immunofluorescence

Journal: Developmental Cell

Article Title: Integrin-Mediated Focal Anchorage Drives Epithelial Zippering during Mouse Neural Tube Closure

doi: 10.1016/j.devcel.2020.01.012

Figure Lengend Snippet: Genetic Ablation of Integrin β1 in SE (A and B) Grhl3 Cre -mediated recombination of the Itgβ1 f/f gene in the SE (diagram in A) as assessed by X-gal staining (B). Grhl3 Cre recombines throughout the SE and in a few dorsal NE cells. Stage: 20 som. (C) Immunostaining in cross-sections confirms loss of integrin β1 in the dorsal SE cells (between arrowheads). (D) Immunostaining on whole-mount embryos confirms focal expression of integrin β1 precisely at the site of neural fold fusion in wild-type (WT) whereas integrin β1 enrichment is lost upon Grhl3 Cre -mediated recombination (mutant). Stages: 24 som, WT and 22 som, Mut. (E) At E10.5, mutant embryos display an open PNP (dotted line), whereas the NT has closed in WT stage-matched littermates. Stages: 33 som, WT and 32 som, Mut. (F and G) Linear regression analysis of PNP length (F) and width (G) at different somite stages in WT (n = 48; length, r = 0.79; width, r 2 = 0.60), Het (n = 37; length, r 2 = 0.69; width, r 2 = 0.66), and Mut (n = 18; length, r 2 = 0.01; width, r 2 = 0.02). Slopes of the regression lines differ significantly between WT and Het and Mut, p < 0.0001. Note cessation of closure in Mut from 20 somites. (H and I) Quantification of NT defects (H) and their appearance at E14.5 (I). The majority of mutants undergo abnormal spinal NT closure, with 56% of E14.5 fetuses exhibiting open spina bifida (I, arrow) and 22% showing tail flexion defects. Fisher’s exact test: p < 0.0001 WT versus Mut. Number of fetuses: n = 38 (WT); n = 16 (Het); and n = 18 (Mut). (J) Open spina bifida lesions are evident perinatally (E18) in mutant fetuses at the lumbo-sacral level (arrow). Scale bars: 500 μm (B) (whole mount); 100 μm (B) (sections, C); 50 μm (B) (sections zoom, C zoom); 500 μm (D); and 2 mm (H) and (I). See also Figure S3 .

Article Snippet: Antibodies : Two different antibodies were used to detect the integrin β1 receptor: a rat monoclonal anti-Integrin β1 (MAB1997) that recognises the full β1 subunit and a rat monoclonal anti-Integrin β1 (BD Biosciences, 553715) that has been reported to recognise the active/ligand-bound form of the β1 subunit ( , ).

Techniques: Staining, Immunostaining, Expressing, Mutagenesis

Journal: Developmental Cell

Article Title: Integrin-Mediated Focal Anchorage Drives Epithelial Zippering during Mouse Neural Tube Closure

doi: 10.1016/j.devcel.2020.01.012

Figure Lengend Snippet: Genetic Ablation of Integrin β1 in the NE (A and B) Pax3 Cre -mediated recombination of the Itgβ1 f/f gene in the dorsal NE (diagram in A) as assessed by whole-mount X-gal (B). Pax3 Cre recombines Itgβ1 in the dorsal NE along the entire body axis of the embryo in both open (II) and closed (I) NT regions. Stage: 24 som. (C) Immunostaining in cross-sections confirms loss of integrin β1 on the basal surface of dorsal neuroepithelial cells (between arrowheads), whereas expression in the SE is unaffected (arrows). (D) Pax3 Cre -mediated recombination of Itgβ1 in the dorsal NE does not abolish focal upregulation of integrin β1 protein at the site of fusion. (E and F) Linear regression analysis of PNP length (E) and width (F) by somite stage in WT (length: n = 46, r 2 = 0.89; width: n = 29, r 2 = 0.51), Het (length: n = 31, r 2 = 0.86; width: n = 15, r 2 = 0.37), and Mut embryos (length: n = 3 4, r 2 = 0.67; n = 23, r 2 = 0.09). Difference in slopes is not significant (ns). Intercept of PNP length differs significantly between WT/Het and Mut; p < 0.0001. (G) At E14.5, 9% of mutant fetuses exhibit open spina bifida and 14% exhibit tail flexion defects. Fisher’s exact test: p = 0.004, WT versus Mut. Open spina bifida also occurs in heterozygotes with similar frequency (9%). Number of embryos: n = 40 (WT), n = 23 (Het), and n = 22 (Mut). Stages: 22 som, WT and 23 som, Mut. Scale bars: 500 μm (B) (left); 200 μm (B) (zoom); 100 μm (B) (sections); 50 μm (B) (section zoom); 50 μm (C); 100 μm (D); and 50 μm (D) (zoom). See also Figure S4 .

Article Snippet: Antibodies : Two different antibodies were used to detect the integrin β1 receptor: a rat monoclonal anti-Integrin β1 (MAB1997) that recognises the full β1 subunit and a rat monoclonal anti-Integrin β1 (BD Biosciences, 553715) that has been reported to recognise the active/ligand-bound form of the β1 subunit ( , ).

Techniques: Immunostaining, Expressing, Mutagenesis

Journal: Developmental Cell

Article Title: Integrin-Mediated Focal Anchorage Drives Epithelial Zippering during Mouse Neural Tube Closure

doi: 10.1016/j.devcel.2020.01.012

Figure Lengend Snippet: Cellular Analysis of Integrin β1 Deletion in the SE Comparison of Grhl3 Cre -targeted mutants and WT embryos for cellular features previously linked to the process of spinal closure. (A) Actomyosin cable originating at the fusion site (arrow) and running along the neural fold edges, at the SE-neuroepithelial interface. No difference is observed between WT and mutants (insets). (B and C) Cellular protrusions (lamellipodia and filopodia) at the site of fusion, as revealed by scanning electron microscopy. Equivalent type of protrusions and distribution observed in WT and mutant embryos (Fisher’s exact test, p > 0.05, n = 4, WT; n = 3, Mut). (D and E) Distribution of cell divisions within the SE as detected by phospho-histone H3 staining (D) with quantification (E). WT and mutants do not differ (mitoses as % of total SE cells, Mann-Whitney Test, p > 0.05, n = 4, WT; n = 4, Mut). (F and G) Programmed cell death in SE as revealed by TUNEL staining at E9.5 (G) (top) and E10.5 (G) (bottom) does not differ at the site of fusion between WT and mutants, although apoptosis is increased by ∼ 25% over the closed NT in mutants (F: two-way ANOVA; fusion site, p > 0.05; closed NT, p = 0.03; n = 3, WT; n = 3, Mut). Scale bars: 100 μm (A), (B), (D), and (G, top); 50 μm (A) (zoom); 10 μm (B) (lower images); and 200 μm (G) (bottom).

Article Snippet: Antibodies : Two different antibodies were used to detect the integrin β1 receptor: a rat monoclonal anti-Integrin β1 (MAB1997) that recognises the full β1 subunit and a rat monoclonal anti-Integrin β1 (BD Biosciences, 553715) that has been reported to recognise the active/ligand-bound form of the β1 subunit ( , ).

Techniques: Electron Microscopy, Mutagenesis, Staining, MANN-WHITNEY, TUNEL Assay

Journal: Developmental Cell

Article Title: Integrin-Mediated Focal Anchorage Drives Epithelial Zippering during Mouse Neural Tube Closure

doi: 10.1016/j.devcel.2020.01.012

Figure Lengend Snippet: Biomechanical and Morphometric Analysis of Integrin β1 Deletion in SE (A–C) Laser ablation of the zippering point (A) with imaging of the lateral recoil of the neural folds (B). WT and mutant embryos do not differ in the amount of neural fold recoil along the PNP axis (C) (linear regression: WT, r 2 = 0.29; Mut, r 2 = 0.33; difference in slopes, p > 0.05; n = 9, WT; n = 7, Mut). Stages: 20–24 som. (D and E) Wound stab assay, as an indicator of mechanical tension in the SE: location along body axis (D, left), typical recoil responses (D, right) and quantification (E). Mut shows a significant increase in recoil compared with WT (Mann-Whitney: p = 0.03; n = 9, WT; n = 9, Mut). Stage: 16–20 som. (F–H) Morphometric analysis of the SE. Dorsal view of SE (F, left) with boxed area enlarged in diagram (F, right). Loss of integrin β1 causes a significant increase in SE cell surface area (G) while rostro-caudal orientation (H) is maintained (Mann-Whitney: median cell area, p = 0.0079; median cell orientation, p > 0.05; n = 5 WT embryos, 884 cells; n = 5 Mut embryos, n = 1,219 cells). Stages: 20–24 som. (I–K) Morphometric analysis of the SE dorsal midline, with cells analysed indicated in grey (I). Loss of integrin β1 causes increased SE cell width (K) but not length (J) (Mann-Whitney: median cell width, p = 0.0004; median cell length, p > 0.05; n = 9 WT embryos, n = 206 cells; n = 6 Mut embryos, n = 181 cells). (L) Immunostaining for integrin β1 in WT embryos at E9.5. Insets: virtual cross-section (bottom left) and dorsal views (top and bottom right). SE cells adopt a semi-rosette configuration at the zippering site, converging on the point of integrin β1 and fibronectin co-expression. Scale bars: 100 μm (A) and (D); 50 μm (I) and (L); and 25 μm (L) (zoom). See also Figure S5 .

Article Snippet: Antibodies : Two different antibodies were used to detect the integrin β1 receptor: a rat monoclonal anti-Integrin β1 (MAB1997) that recognises the full β1 subunit and a rat monoclonal anti-Integrin β1 (BD Biosciences, 553715) that has been reported to recognise the active/ligand-bound form of the β1 subunit ( , ).

Techniques: Imaging, Mutagenesis, MANN-WHITNEY, Immunostaining, Expressing

Journal: Developmental Cell

Article Title: Integrin-Mediated Focal Anchorage Drives Epithelial Zippering during Mouse Neural Tube Closure

doi: 10.1016/j.devcel.2020.01.012

Figure Lengend Snippet: Cellular Arrangement at the Zippering Point in Static- and Live-Imaged Embryos (A) At E9.5, SE cells 1–3 form a semi-rosette at the zippering point in WT embryos, as revealed by E-cadherin (Ecad) staining (top) and 3D cell reconstruction (bottom). Mutant embryos do not exhibit a regular semi-rosette. (B) Semi-rosettes contain on average 7 cells in WT and 3–4 cells in mutants (Mann-Whitney: p = 0.0002; embryos: n = 10 WT; n = 6 Mut). (C and D) At E9.5 (19–25 som), SE cells 1–3 display a wedge-shaped morphology (junction length ratio ≈ 0) due to extreme shortening of their proximal junctions (C)–(D) (WT). Cells bordering the open PNP (cells 4–7) exhibit a more “rectangular” morphology (junction length ratio ≈ 1; C–D [WT]). Integrin β1-deficient SE cells at the site of fusion (cells 2–3) fail to shorten proximal junctions while cells 4–7 bordering the open PNP maintain a morphology similar to WT (C)–(D) (Mut) (2-way ANOVA, post-hoc Bonferroni test; cell 1: p > 0.05; cells 2–3: p < 0.001; cell 4: p = 0.028; cells 5–7: p > 0.05; embryos: n = 10 WT (137 cells); n = 6 Mut [72 cells]). (E–G) At E9.0 (10–18 som), WT SE cells 1–2 at the site of fusion form a smaller semi-rosette than at E9.5. Mutant SE cells 1–2 display a semi-rosette but with a less prominent wedge-shaped morphology (E). Junction length ratio does not differ from WT (F, 2-way ANOVA, post-hoc Bonferroni test; p > 0.05; embryos: n = 8 WT (112 cells); n = 7 Mut [98 cells]). Each semi-rosette contains on average 3–4 cells both in WT and mutants (G, Mann-Whitney: p > 0.05; embryos: n = 8 WT; n = 7 Mut). (H) Live imaging SE cell dynamics during zippering. Individual cells (indicated by colors) shorten their proximal junctions over time to form the semi-rosette configuration and then exit the zippering point rostrally, with further cell elongation. (I and J) Model of semi-rosette formation and zippering propagation. SE cells upregulate integrin α5β1 at the fusion site, with coordinated adhesion to fibronectin (SE = Fn1) causing proximal junctions to shorten forming a semi-rosette (I). Opposing junctions are brought into close proximity (J), enabling cross-midline junction formation at the site of shared basal adhesion (Se = Fn1 = SE). This propagates zippering forward with novel cell-cell junction formation (SE = SE). Scale bars: 50 μm (A) and (E). See also Figure S5 and , , , , and .

Article Snippet: Antibodies : Two different antibodies were used to detect the integrin β1 receptor: a rat monoclonal anti-Integrin β1 (MAB1997) that recognises the full β1 subunit and a rat monoclonal anti-Integrin β1 (BD Biosciences, 553715) that has been reported to recognise the active/ligand-bound form of the β1 subunit ( , ).

Techniques: Staining, Mutagenesis, MANN-WHITNEY, Imaging

Journal: Developmental Cell

Article Title: Integrin-Mediated Focal Anchorage Drives Epithelial Zippering during Mouse Neural Tube Closure

doi: 10.1016/j.devcel.2020.01.012

Figure Lengend Snippet:

Article Snippet: Antibodies : Two different antibodies were used to detect the integrin β1 receptor: a rat monoclonal anti-Integrin β1 (MAB1997) that recognises the full β1 subunit and a rat monoclonal anti-Integrin β1 (BD Biosciences, 553715) that has been reported to recognise the active/ligand-bound form of the β1 subunit ( , ).

Techniques: TUNEL Assay, Staining, In Situ, Software