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: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: Conserved residues in the C-terminal region of kindlin-2 support αIIbβ3 activation in αIIbβ3-CHO cells. A, alignment of C-terminal sequences of human K2 and Fit1, the K2 homolog of D. melanogaster. B and C, αIIbβ3-CHO cells were transiently transfected with plasmids encoding EGFP-fused FL, wild-type K2 or its mutants and DsRed talin-H, and activation of αIIbβ3 integrin was determined at 24 h by flow cytometry by staining the cells with the activation-specific antibody PAC-1 (see “Experimental procedures”). Error bars, S.D. Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S1.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Activation Assay, Transfection, Flow Cytometry, Staining

Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: The kindlin-2 C-terminal segment supports integrin function in HEL megakaryotic and RAW 264.7 macrophage-like cells. A, HEL cells were transiently transfected with plasmids encoding EGFP-fused K2 or its mutants and DsRed talin-H, and the extent of αIIbβ3 activation in DsRed and EGFP double-positive cells was quantified by flow cytometry with the activation-specific antibody PAC-1 (see “Experimental procedures”). The experiments were performed three times. Error bars, S.D. The total αIIbβ3 expression, measured with an mAb unaffected by the activation status of the receptor, in the presence and absence of the various K2s with or without talin-H was unaffected. The expression levels of the K2 mutants were similar. B, HEL cells were transiently transfected with plasmids encoding EGFP alone and EGFP-K2 constructs. After 24 h, EGFP expression levels in HEL cells were determined by flow cytometry; transfection efficiency was 70–80%. The transfected HEL cells were treated with PMA and allowed to adhere to fibrinogen-coated coverslips, and cell spreading was measured after 30 min. The adherent cells were fixed and stained with Alexa 568 phalloidin, and the areas of cells were measured using ImageJ software; 300 cells were quantified per construct. Error bars, S.D. (p < 0.001). C, mouse RAW 264.7 cells were transiently transfected with plasmids encoding EGFP alone or EGFP-K2 constructs. The transfected cells were stained with 9EG7 monoclonal antibody to assess β1 integrin activation. Flow cytometry was used to measure 9EG7 binding. Error bars, S.E. of three independent experiments (p < 0.001). Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S2.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Transfection, Activation Assay, Flow Cytometry, Expressing, Construct, Staining, Software, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: The C-terminal segment of kindlin-1 is necessary to maintain the co-activator function of K1 in talin-H mediated integrin activation in αIIbβ3-CHO cells. A, alignment of C-terminal sequences of human K1 and human K2. B, αIIbβ3-CHO cells were transiently transfected with plasmids encoding EGFP-fused K1, wild type or its mutants, and DsRed talin-H. After 24 h, activation of the integrin was quantified by flow cytometry as in Fig. 1. Error bars, S.D. Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S3.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Activation Assay, Transfection, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: The C-terminal segment of kindlin-3 is dispensable for K3 functions. A, alignment of C-terminal sequences of human K2 and K3; B, αIIbβ3 integrin co-activation analysis using αIIbβ3-CHO cells transfected with EGFP-fused wild-type K3 or K3 chimera and talin-H; the activation of the integrin was determined after 24 h by flow cytometry with the activation-specific antibody PAC-1 (see “Experimental procedures”). Error bars, S.D. Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S4.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Activation Assay, Transfection, 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: 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: Molecular Biology of the Cell

Article Title: β1A Integrin Is a Master Regulator of Invadosome Organization and Function

doi: 10.1091/mbc.E10-07-0580

Figure Lengend Snippet: Extracellular matrix sensing by β1 and β3 integrins controls invadosome formation and localization. (A) Most SrcYF-expressing cells formed invadosome rosettes (visualized by F-actin staining, in green) only on the adhesive surface (gelatin-tetramethylrhodamine B isothiocyanate [TRITC], in red, mixed with vitronectin) and not on antiadhesive areas (Pluronic F127, black areas). (B) Invadosome rosettes stained by phalloidin-TRITC show higher affinity for vitronectin-FITC (light gray bands), sensed by members of the β3 integrin family, than for fibronectin (black bands), sensed by members of both the β1 and β3 integrin families. (C) The β1 and β3 integrins show distinct patterns of localization, observed in MEF-SrcYF cells. β3 (in green in merge) highly colocalized with F-actin, whereas β1 staining is limited to the rosette periphery. (D) 9EG7 antibody β1 staining is specific of the activated form of the integrin. Antibody against activated form of β1 directly conjugated to FITC (9EG7-FITC) was added externally and localized around the invadosome visualized by cortactin-mRFP (in red in merge) expressed in live MEF-SrcYF cells. Bars, 5 μm (A and B), 4 μm (C), and 2 μm (D).

Article Snippet: Antibodies for immunoblotting and immunofluorescence were obtained from the following commercial sources: rabbit anti-phospho-Tyr 418 Src and anti-phospho-Tyr397 FAK (Invitrogen, Carlsbad, CA), mouse anti-Src (GD11; Millipore, Billerica, MA), mouse anti-actin (Sigma-Aldrich, St. Louis, MO), mouse anti-paxillin (BD Biosciences, Franklin Lakes, NJ), mouse anti-human β1 integrin (clone 4B7R; Becton Dickinson, le Pont de Claix, France), rat anti-mouse β1 integrin (MB1.2; Millipore), and rat anti-mouse β3 integrin (clone LucA5; Emfret Analytics, Würzburg, Germany).

Techniques: Expressing, Staining

Journal: Molecular Biology of the Cell

Article Title: β1A Integrin Is a Master Regulator of Invadosome Organization and Function

doi: 10.1091/mbc.E10-07-0580

Figure Lengend Snippet: β1A, and not β3, integrin is essential for invadosome formation. (A) β3 is not essential for invadosome formation and self-assembly into rosettes, visualized by F-actin (in red in merge) and phospho-Y397-FAK (in blue in merge) staining, which occurs in both MEF-SrcYF β3 +/+ or −/− cells. (B) In contrast, β1 depletion in MEF-SrcYF β1 LoxP/LoxP expressing the CRE recombinase for 96 h resulted in the disappearance of isolated invadosomes or in rosettes probed by phalloidin staining. (C) Quantification of the percentage of cells forming invadosomes reveals that almost 95% of MEF-SrcYF β1 LoxP/LoxP treated with CRE recombinase did not form this structure 4 d after infection (n = 650 counted cells/condition). (D) Quantification by qPCR shows an average decrease of 95% in the level of β1 mRNA 96 h post-CRE treatment. Bars, 3 μm (A) and 10 μm (B).

Article Snippet: Antibodies for immunoblotting and immunofluorescence were obtained from the following commercial sources: rabbit anti-phospho-Tyr 418 Src and anti-phospho-Tyr397 FAK (Invitrogen, Carlsbad, CA), mouse anti-Src (GD11; Millipore, Billerica, MA), mouse anti-actin (Sigma-Aldrich, St. Louis, MO), mouse anti-paxillin (BD Biosciences, Franklin Lakes, NJ), mouse anti-human β1 integrin (clone 4B7R; Becton Dickinson, le Pont de Claix, France), rat anti-mouse β1 integrin (MB1.2; Millipore), and rat anti-mouse β3 integrin (clone LucA5; Emfret Analytics, Würzburg, Germany).

Techniques: Staining, Expressing, Isolation, Infection

Journal: Molecular Biology of the Cell

Article Title: β1A Integrin Is a Master Regulator of Invadosome Organization and Function

doi: 10.1091/mbc.E10-07-0580

Figure Lengend Snippet: Activation of β1 stimulates invadosome autoassembly. (A) β1 depletion in pOBL-SrcYF induced the disappearance of invadosomes, shown by phalloidin staining of F-actin. Bar, 5 μm. (B) β1 depletion does not affect Src activation. Lysates of pOBL-SrcYF β1 +/+ and −/− cells were probed by Western blotting for phospho-SrcY416, a marker of Src activation, total Src, and actin. (C) Amino-acyl sequence of the cytoplasmic domain of β1A integrin and the location of the main mutation that activates this integrin. (D) Expression of the preactivated mutants of β1 (β1 D759A) in pOBL-SrcYF β1 −/− cells dramatically increases the number of invadosome rosettes per airy unit, whereas β1WT mutant rescue the rosette number up to the control level.

Article Snippet: Antibodies for immunoblotting and immunofluorescence were obtained from the following commercial sources: rabbit anti-phospho-Tyr 418 Src and anti-phospho-Tyr397 FAK (Invitrogen, Carlsbad, CA), mouse anti-Src (GD11; Millipore, Billerica, MA), mouse anti-actin (Sigma-Aldrich, St. Louis, MO), mouse anti-paxillin (BD Biosciences, Franklin Lakes, NJ), mouse anti-human β1 integrin (clone 4B7R; Becton Dickinson, le Pont de Claix, France), rat anti-mouse β1 integrin (MB1.2; Millipore), and rat anti-mouse β3 integrin (clone LucA5; Emfret Analytics, Würzburg, Germany).

Techniques: Activation Assay, Staining, Western Blot, Marker, Sequencing, Mutagenesis, Expressing

Journal: Molecular Biology of the Cell

Article Title: β1A Integrin Is a Master Regulator of Invadosome Organization and Function

doi: 10.1091/mbc.E10-07-0580

Figure Lengend Snippet: PKC regulates invadosome autoassembly by phosphorylating Ser785 of β1A integrins. (A) PKC activation by a 60-min treatment of pOBL-SrcYF cells with 2 μM PMA induces a massive increase in invadosome rosette autoassembly, visualized by phalloidin staining. (B) Extracted images from time series (in minutes) from representative observations of pOBL-SrcYF cells expressing GFP-actin and treated with either dimethyl sulfoxide (DMSO; control), the PKC activator PMA (2 μM), and the PKC inhibitor BIM (5 mM). PKC activity regulates the dynamics and maintenance of the invadosome autoassembly state. (C) Amino-acyl sequence of the cytoplasmic domain of β1A integrin and the location of the main PKC targets. (D) Quantification of invadosomes per Airy unit (AU) shows that a mutation mimicking a constitutive phosphorylated form of Ser785 (Asp or Glu) dramatically increases the formation of rosettes in pOBL-SrcYF β1 −/− cells. The nonphosphorylatable mutant of β1 at this site (S785A) has no effect on rosette formation. (E) The number of rosettes/AU was quantified in pOBL-SrcYF cells treated with 2 μM PMA or simultaneously with PMA and 5 mM BIM for 60 min. The inhibitory effect of BIM was determined by calculating the percent inhibition of invadosome rosette formation. Mutants mimicking a constitutively phosphorylated Ser785 (S785D and S785E) show only 50% inhibition after BIM treatment, indicating that this residue on β1 is a major target of PKC in the regulation of invadosome autoassembly. Bars, 20 μm (A) and 5 μm (B).

Article Snippet: Antibodies for immunoblotting and immunofluorescence were obtained from the following commercial sources: rabbit anti-phospho-Tyr 418 Src and anti-phospho-Tyr397 FAK (Invitrogen, Carlsbad, CA), mouse anti-Src (GD11; Millipore, Billerica, MA), mouse anti-actin (Sigma-Aldrich, St. Louis, MO), mouse anti-paxillin (BD Biosciences, Franklin Lakes, NJ), mouse anti-human β1 integrin (clone 4B7R; Becton Dickinson, le Pont de Claix, France), rat anti-mouse β1 integrin (MB1.2; Millipore), and rat anti-mouse β3 integrin (clone LucA5; Emfret Analytics, Würzburg, Germany).

Techniques: Activation Assay, Staining, Expressing, Activity Assay, Sequencing, Mutagenesis, Inhibition