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Developmental Studies Hybridoma Bank human β1 integrin

E0771 cells have reduced expression of the high-affinity reovirus attachment receptor JAM-A. E0771 and L929 cells were harvested using CellStripper, fixed with 4% paraformaldehyde (PFA), and analyzed by flow cytometry following immunostaining with monoclonal antibodies specific for SNA, JAM-A, or <t>β1</t> <t>integrin.</t> Representative histograms (left) and corresponding quantification (right) show cell surface expression of ( A ) α2,6-linked sialic acids detected using fluorescently labeled SNA lectin in neuraminidase-treated (+) or untreated (−) cells, ( B ) JAM-A detected using murine JAM-A-specific antibodies (+) compared with isotype (Iso) controls, and ( C ) β1 integrin detected using β1 integrin-specific primary antibodies (+) compared with Iso controls. MFIs were normalized to the corresponding negative controls (neuraminidase-treated or isotype-stained cells) from a representative L929 experiment. Data represent mean ± SD ( n = 3). Statistical significance was determined by the two-way ANOVA with Tukey’s multiple comparisons test in GraphPad Prism v10.4 (ns = P > 0.05; * P < 0.05; ** P < 0.005; *** P < 0.001; and **** P < 0.0001).

Human β1 Integrin, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Article Title: Enhanced sialic acid engagement at physiological temperatures by reovirus σ1 mutants facilitates infection of breast cancer cells with low levels of high-affinity receptors

Journal: Journal of Virology

doi: 10.1128/jvi.00074-26

Figure Legend Snippet: E0771 cells have reduced expression of the high-affinity reovirus attachment receptor JAM-A. E0771 and L929 cells were harvested using CellStripper, fixed with 4% paraformaldehyde (PFA), and analyzed by flow cytometry following immunostaining with monoclonal antibodies specific for SNA, JAM-A, or β1 integrin. Representative histograms (left) and corresponding quantification (right) show cell surface expression of ( A ) α2,6-linked sialic acids detected using fluorescently labeled SNA lectin in neuraminidase-treated (+) or untreated (−) cells, ( B ) JAM-A detected using murine JAM-A-specific antibodies (+) compared with isotype (Iso) controls, and ( C ) β1 integrin detected using β1 integrin-specific primary antibodies (+) compared with Iso controls. MFIs were normalized to the corresponding negative controls (neuraminidase-treated or isotype-stained cells) from a representative L929 experiment. Data represent mean ± SD ( n = 3). Statistical significance was determined by the two-way ANOVA with Tukey’s multiple comparisons test in GraphPad Prism v10.4 (ns = P > 0.05; * P < 0.05; ** P < 0.005; *** P < 0.001; and **** P < 0.0001).

Techniques Used: Expressing, Flow Cytometry, Immunostaining, Bioprocessing, Labeling, Staining

Mutations in the sialic acid-binding domain permit high-affinity, receptor-independent attachment by increasing binding to sialic acids at physiological temperature (37°C). ( A ) Representative western blot analysis of outer capsid proteins µ1C and σ3, detected using anti-reovirus polyclonal serum. ( B and C ) L929 ( B ) and E0771 ( C ) cells were treated with PBS or neuraminidase (+neuraminidase) for 1 h at 37°C to deplete cell surface sialic acids. Viruses indicated in the legend were incubated with cells for 1 h at 4°C or 37°C in the presence of NH 4 Cl, washed, and cells processed for flow cytometric analysis using σ3-specific antibodies. MFI reflects the level of cell-associated virus particles. ( D ) Virus-cell association was measured as in panel C , without neuraminidase treatment, using parental U937 cells or U937 cells deficient in sialic acids (U937-Sia - ). ( E ) Flow cytometric detection of α2,6-linked sialic acids on RBCs using fluorescently labeled SNA lectin. Representative histograms (left) show unstained RBCs (light gray), SNA-stained RBCs (red), and SNA-stained H1299 cells (dark gray). n = 3. ( F ) Flow cytometric detection of JAM-A on RBCs using a primary/secondary antibody system specific for hJAM-A. Representative histograms (left) show RBCs with secondary antibody only (light gray), primary/secondary-stained RBCs (red), and E0771+JAM cells (dark gray). n = 3. ( G ) Flow cytometric detection of β1 integrins on RBCs using primary/secondary antibodies specific for human β1 integrin. Representative histograms (left) show RBCs with secondary antibody only (light gray), primary/secondary-stained RBCs (red), and H1299 cells (dark gray). n = 3. ( H ) RBCs were incubated with particle-normalized T3D PL at serial dilutions starting at 1.9 × 10 5 particles for 1 h at 4°C or 37°C. Unbound virions were removed by PBS washes prior to fixation and immunostaining for outer capsid proteins, followed by flow cytometric analysis. n = 3. ( I–K ) RBCs were incubated with particle-normalized T3D PL or variant viruses at serial dilutions starting at 1.9 × 10 5 particles for 1 h at 4°C ( I and J ) or 37°C ( I and K ). Following removal of unbound virions by PBS washes, cells were fixed, immunostained for outer capsid proteins, and analyzed by flow cytometry. Absolute MFI values ( I ) were used to calculate the AUC for each virus across all independent experiments, normalized to T3D PL at the corresponding temperature for each independent experiment ( n = 3–5). ( L ) Levels of σ1 per virion for full-length T3D PLσ1-G196R were assessed by agarose gel electrophoresis (top) and quantitative serial dilution-based western blot analysis using anti-σ3 and anti-µ1 monoclonal antibodies and anti-σ1 tail polyclonal antibodies (middle). Bottom: relative average σ1 per virion calculated relative to T3D PL from five independent virus preparations based on σ1 to (σ3 + µ1) protein ratios determined by western blot analysis. Data represent mean ± SD. Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test ( E, J, and K ) or the paired t -test ( F, G, and H ) in GraphPad Prism v10.4. (ns = P > 0.05; * P < 0.05; ** P < 0.005; *** P < 0.001; and **** P < 0.0001).

Figure Legend Snippet: Mutations in the sialic acid-binding domain permit high-affinity, receptor-independent attachment by increasing binding to sialic acids at physiological temperature (37°C). ( A ) Representative western blot analysis of outer capsid proteins µ1C and σ3, detected using anti-reovirus polyclonal serum. ( B and C ) L929 ( B ) and E0771 ( C ) cells were treated with PBS or neuraminidase (+neuraminidase) for 1 h at 37°C to deplete cell surface sialic acids. Viruses indicated in the legend were incubated with cells for 1 h at 4°C or 37°C in the presence of NH 4 Cl, washed, and cells processed for flow cytometric analysis using σ3-specific antibodies. MFI reflects the level of cell-associated virus particles. ( D ) Virus-cell association was measured as in panel C , without neuraminidase treatment, using parental U937 cells or U937 cells deficient in sialic acids (U937-Sia - ). ( E ) Flow cytometric detection of α2,6-linked sialic acids on RBCs using fluorescently labeled SNA lectin. Representative histograms (left) show unstained RBCs (light gray), SNA-stained RBCs (red), and SNA-stained H1299 cells (dark gray). n = 3. ( F ) Flow cytometric detection of JAM-A on RBCs using a primary/secondary antibody system specific for hJAM-A. Representative histograms (left) show RBCs with secondary antibody only (light gray), primary/secondary-stained RBCs (red), and E0771+JAM cells (dark gray). n = 3. ( G ) Flow cytometric detection of β1 integrins on RBCs using primary/secondary antibodies specific for human β1 integrin. Representative histograms (left) show RBCs with secondary antibody only (light gray), primary/secondary-stained RBCs (red), and H1299 cells (dark gray). n = 3. ( H ) RBCs were incubated with particle-normalized T3D PL at serial dilutions starting at 1.9 × 10 5 particles for 1 h at 4°C or 37°C. Unbound virions were removed by PBS washes prior to fixation and immunostaining for outer capsid proteins, followed by flow cytometric analysis. n = 3. ( I–K ) RBCs were incubated with particle-normalized T3D PL or variant viruses at serial dilutions starting at 1.9 × 10 5 particles for 1 h at 4°C ( I and J ) or 37°C ( I and K ). Following removal of unbound virions by PBS washes, cells were fixed, immunostained for outer capsid proteins, and analyzed by flow cytometry. Absolute MFI values ( I ) were used to calculate the AUC for each virus across all independent experiments, normalized to T3D PL at the corresponding temperature for each independent experiment ( n = 3–5). ( L ) Levels of σ1 per virion for full-length T3D PLσ1-G196R were assessed by agarose gel electrophoresis (top) and quantitative serial dilution-based western blot analysis using anti-σ3 and anti-µ1 monoclonal antibodies and anti-σ1 tail polyclonal antibodies (middle). Bottom: relative average σ1 per virion calculated relative to T3D PL from five independent virus preparations based on σ1 to (σ3 + µ1) protein ratios determined by western blot analysis. Data represent mean ± SD. Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test ( E, J, and K ) or the paired t -test ( F, G, and H ) in GraphPad Prism v10.4. (ns = P > 0.05; * P < 0.05; ** P < 0.005; *** P < 0.001; and **** P < 0.0001).

Techniques Used: Binding Assay, Western Blot, Incubation, Virus, Labeling, Staining, Immunostaining, Variant Assay, Flow Cytometry, Agarose Gel Electrophoresis, Serial Dilution, Bioprocessing

Mutational enhancement of σ1-mediated receptor binding and structural basis of sialic acid interaction. ( A ) Model depiction of relative binding strengths deduced from experimental mean AUCs between the sialic acid-binding domain (orange) to sialic acids (dark gray, Sia), the JAM-A-binding domain (circular head of σ1) to JAM-A (black), and the RGD domain (red) to β-integrin (light gray, βInt). Where “~” is indicated, relative binding strength was deduced by subtracting the total binding measured in the JAM-A-deficient condition from the domain-specific binding strength. ( B ) Structural models of the T3D PL σ1 body domain were generated using UCSF ChimeraX (v1.9). Wild-type (bordered) and mutant σ1 structures were created by introducing identified substitutions. Predicted hydrogen bonds with α2,3-linked sialic acid (PDB: 3S6X) were assessed using default cutoffs; a representative G196R rotamer shows novel hydrogen bonds between the arg196 and sialic acid (red arrow). ( C ) Amino acid sequence alignment of the σ1 body domain encompassing the sialic acid-binding pocket (NCBI). Mutations identified through passage (G196R, T193M, and N206H) are annotated alongside known sialic acid-binding residues (N198, R202, and P204).

Figure Legend Snippet: Mutational enhancement of σ1-mediated receptor binding and structural basis of sialic acid interaction. ( A ) Model depiction of relative binding strengths deduced from experimental mean AUCs between the sialic acid-binding domain (orange) to sialic acids (dark gray, Sia), the JAM-A-binding domain (circular head of σ1) to JAM-A (black), and the RGD domain (red) to β-integrin (light gray, βInt). Where “~” is indicated, relative binding strength was deduced by subtracting the total binding measured in the JAM-A-deficient condition from the domain-specific binding strength. ( B ) Structural models of the T3D PL σ1 body domain were generated using UCSF ChimeraX (v1.9). Wild-type (bordered) and mutant σ1 structures were created by introducing identified substitutions. Predicted hydrogen bonds with α2,3-linked sialic acid (PDB: 3S6X) were assessed using default cutoffs; a representative G196R rotamer shows novel hydrogen bonds between the arg196 and sialic acid (red arrow). ( C ) Amino acid sequence alignment of the σ1 body domain encompassing the sialic acid-binding pocket (NCBI). Mutations identified through passage (G196R, T193M, and N206H) are annotated alongside known sialic acid-binding residues (N198, R202, and P204).

Techniques Used: Binding Assay, Generated, Mutagenesis, Sequencing

Purification:

Article Title: Molecular mechanisms of neural crest cell attachment and migration on types I and IV collagen.
Article Snippet: .. CSAT and JG22-producing hybridoma cell lines were purchased from the Developmental Studies Hybridoma Bank (Iowa City, IW) and the IgGs were purified by chromatography on Protein A-Sepharose (Pharmacia). ..

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Journal: Journal of Virology

Article Title: Enhanced sialic acid engagement at physiological temperatures by reovirus σ1 mutants facilitates infection of breast cancer cells with low levels of high-affinity receptors

doi: 10.1128/jvi.00074-26

Figure Lengend Snippet: E0771 cells have reduced expression of the high-affinity reovirus attachment receptor JAM-A. E0771 and L929 cells were harvested using CellStripper, fixed with 4% paraformaldehyde (PFA), and analyzed by flow cytometry following immunostaining with monoclonal antibodies specific for SNA, JAM-A, or β1 integrin. Representative histograms (left) and corresponding quantification (right) show cell surface expression of ( A ) α2,6-linked sialic acids detected using fluorescently labeled SNA lectin in neuraminidase-treated (+) or untreated (−) cells, ( B ) JAM-A detected using murine JAM-A-specific antibodies (+) compared with isotype (Iso) controls, and ( C ) β1 integrin detected using β1 integrin-specific primary antibodies (+) compared with Iso controls. MFIs were normalized to the corresponding negative controls (neuraminidase-treated or isotype-stained cells) from a representative L929 experiment. Data represent mean ± SD ( n = 3). Statistical significance was determined by the two-way ANOVA with Tukey’s multiple comparisons test in GraphPad Prism v10.4 (ns = P > 0.05; * P < 0.05; ** P < 0.005; *** P < 0.001; and **** P < 0.0001).

Article Snippet: To quantify cell-surface receptor expression, cells were incubated with monoclonal antibodies specific for murine JAM-A (clone BV11, Millipore Sigma), murine β1 integrin (eBioscience), human JAM-A (CSTEM27, Thermo Fisher Scientific), or human β1 integrin (clone P5D2, DSHB).

Techniques: Expressing, Flow Cytometry, Immunostaining, Bioprocessing, Labeling, Staining

Journal: Journal of Virology

Article Title: Enhanced sialic acid engagement at physiological temperatures by reovirus σ1 mutants facilitates infection of breast cancer cells with low levels of high-affinity receptors

doi: 10.1128/jvi.00074-26

Figure Lengend Snippet: Mutations in the sialic acid-binding domain permit high-affinity, receptor-independent attachment by increasing binding to sialic acids at physiological temperature (37°C). ( A ) Representative western blot analysis of outer capsid proteins µ1C and σ3, detected using anti-reovirus polyclonal serum. ( B and C ) L929 ( B ) and E0771 ( C ) cells were treated with PBS or neuraminidase (+neuraminidase) for 1 h at 37°C to deplete cell surface sialic acids. Viruses indicated in the legend were incubated with cells for 1 h at 4°C or 37°C in the presence of NH 4 Cl, washed, and cells processed for flow cytometric analysis using σ3-specific antibodies. MFI reflects the level of cell-associated virus particles. ( D ) Virus-cell association was measured as in panel C , without neuraminidase treatment, using parental U937 cells or U937 cells deficient in sialic acids (U937-Sia - ). ( E ) Flow cytometric detection of α2,6-linked sialic acids on RBCs using fluorescently labeled SNA lectin. Representative histograms (left) show unstained RBCs (light gray), SNA-stained RBCs (red), and SNA-stained H1299 cells (dark gray). n = 3. ( F ) Flow cytometric detection of JAM-A on RBCs using a primary/secondary antibody system specific for hJAM-A. Representative histograms (left) show RBCs with secondary antibody only (light gray), primary/secondary-stained RBCs (red), and E0771+JAM cells (dark gray). n = 3. ( G ) Flow cytometric detection of β1 integrins on RBCs using primary/secondary antibodies specific for human β1 integrin. Representative histograms (left) show RBCs with secondary antibody only (light gray), primary/secondary-stained RBCs (red), and H1299 cells (dark gray). n = 3. ( H ) RBCs were incubated with particle-normalized T3D PL at serial dilutions starting at 1.9 × 10 5 particles for 1 h at 4°C or 37°C. Unbound virions were removed by PBS washes prior to fixation and immunostaining for outer capsid proteins, followed by flow cytometric analysis. n = 3. ( I–K ) RBCs were incubated with particle-normalized T3D PL or variant viruses at serial dilutions starting at 1.9 × 10 5 particles for 1 h at 4°C ( I and J ) or 37°C ( I and K ). Following removal of unbound virions by PBS washes, cells were fixed, immunostained for outer capsid proteins, and analyzed by flow cytometry. Absolute MFI values ( I ) were used to calculate the AUC for each virus across all independent experiments, normalized to T3D PL at the corresponding temperature for each independent experiment ( n = 3–5). ( L ) Levels of σ1 per virion for full-length T3D PLσ1-G196R were assessed by agarose gel electrophoresis (top) and quantitative serial dilution-based western blot analysis using anti-σ3 and anti-µ1 monoclonal antibodies and anti-σ1 tail polyclonal antibodies (middle). Bottom: relative average σ1 per virion calculated relative to T3D PL from five independent virus preparations based on σ1 to (σ3 + µ1) protein ratios determined by western blot analysis. Data represent mean ± SD. Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test ( E, J, and K ) or the paired t -test ( F, G, and H ) in GraphPad Prism v10.4. (ns = P > 0.05; * P < 0.05; ** P < 0.005; *** P < 0.001; and **** P < 0.0001).

Techniques: Binding Assay, Western Blot, Incubation, Virus, Labeling, Staining, Immunostaining, Variant Assay, Flow Cytometry, Agarose Gel Electrophoresis, Serial Dilution, Bioprocessing

Journal: Journal of Virology

Article Title: Enhanced sialic acid engagement at physiological temperatures by reovirus σ1 mutants facilitates infection of breast cancer cells with low levels of high-affinity receptors

doi: 10.1128/jvi.00074-26

Figure Lengend Snippet: Mutational enhancement of σ1-mediated receptor binding and structural basis of sialic acid interaction. ( A ) Model depiction of relative binding strengths deduced from experimental mean AUCs between the sialic acid-binding domain (orange) to sialic acids (dark gray, Sia), the JAM-A-binding domain (circular head of σ1) to JAM-A (black), and the RGD domain (red) to β-integrin (light gray, βInt). Where “~” is indicated, relative binding strength was deduced by subtracting the total binding measured in the JAM-A-deficient condition from the domain-specific binding strength. ( B ) Structural models of the T3D PL σ1 body domain were generated using UCSF ChimeraX (v1.9). Wild-type (bordered) and mutant σ1 structures were created by introducing identified substitutions. Predicted hydrogen bonds with α2,3-linked sialic acid (PDB: 3S6X) were assessed using default cutoffs; a representative G196R rotamer shows novel hydrogen bonds between the arg196 and sialic acid (red arrow). ( C ) Amino acid sequence alignment of the σ1 body domain encompassing the sialic acid-binding pocket (NCBI). Mutations identified through passage (G196R, T193M, and N206H) are annotated alongside known sialic acid-binding residues (N198, R202, and P204).

Techniques: Binding Assay, Generated, Mutagenesis, Sequencing

$Desialylation results in the activation of the Hippo pathway. A, MDA-MB-231 cells were pretreated with different doses of sialidase for 3 h; then, the cell membrane fractions were immunoblotted with SNA (recognizing α2,6-sialylated proteins) and ConA (an α-mannose/α-glucose-binding lectin) lectins or blotted with anti-integrin β1 antibody. B, to further determine the change of sialylation on the cell surface after sialidase treatment, the indicated cells were incubated with biotin-conjugated MAA (recognizing 2,3-sialylated proteins, dotted line ), biotin-conjugated SNA ( bold line ), or without ( gray shadow ) lectin, followed by incubation with appropriate Alexa Flour 647 conjugate and subjected to flow cytometry. C, MDA-MB-231 cells were treated as described in ( A ), and then the cell lysates were immunoblotted with anti-p-YAP S127, anti-YAP, anti-p-LATS1 T1079, anti-LATS1, and anti-GAPDH antibodies. The relative ratios (phospho-YAP and phospho-LATS1 versus YAP and LATS1, respectively) are presented as the mean ± SD ( n = 3 biological replicates, ∗∗, p < 0.01, ∗∗∗, p < 0.001 is determined by two-tail unpaired t test). SNA, Sambucus nigra; MAA, Maackia amurensis agglutinin; ConA, Concanavalin A; LATS, large tumor suppressor kinase; YAP, yes-associated protein.$

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: Desialylation results in the activation of the Hippo pathway. A, MDA-MB-231 cells were pretreated with different doses of sialidase for 3 h; then, the cell membrane fractions were immunoblotted with SNA (recognizing α2,6-sialylated proteins) and ConA (an α-mannose/α-glucose-binding lectin) lectins or blotted with anti-integrin β1 antibody. B, to further determine the change of sialylation on the cell surface after sialidase treatment, the indicated cells were incubated with biotin-conjugated MAA (recognizing 2,3-sialylated proteins, dotted line ), biotin-conjugated SNA ( bold line ), or without ( gray shadow ) lectin, followed by incubation with appropriate Alexa Flour 647 conjugate and subjected to flow cytometry. C, MDA-MB-231 cells were treated as described in ( A ), and then the cell lysates were immunoblotted with anti-p-YAP S127, anti-YAP, anti-p-LATS1 T1079, anti-LATS1, and anti-GAPDH antibodies. The relative ratios (phospho-YAP and phospho-LATS1 versus YAP and LATS1, respectively) are presented as the mean ± SD ( n = 3 biological replicates, ∗∗, p < 0.01, ∗∗∗, p < 0.001 is determined by two-tail unpaired t test). SNA, Sambucus nigra; MAA, Maackia amurensis agglutinin; ConA, Concanavalin A; LATS, large tumor suppressor kinase; YAP, yes-associated protein.

Article Snippet: The experiments were performed using the following antibodies: Rabbit antibodies against p-YAP(S127) (#13008S), p-LATS1(T1079) (#8654S), LATS1 (#3477S), p-Src(Y416) (#2101S), p-FAK(Y397) (#8556S), FAK (#3285S), EGFR (#4267S), p-EGFR(Y1068) (#3777S), and integrin β1 (#9699S) were from Cell Signaling Technology; mouse mAb against GAPDH (#sc-365062), and β-actin (#sc-47778) were from Santa Cruz Biotechnology; mouse mAb against integrin α5 (610633) was from BD Biosciences; rabbit pAbs against LPAR4 (22165-1-AP) and mouse mAb against YAP (66900-1-Ig) were obtained from Proteintech; rabbit pAb against ST3GAL4 (NBP1-69565) was obtained from Novus Biologicals; mouse mAbs against FLAG (clone M2, #F3165) and Src (clone GD11, #05-184) were from Sigma; goat pAb against ST6GAL1 (AF5924) was from R&D Systems; mouse mAb against integrin β1 (P5D2) was from Developmental Studies Hybridoma Bank.

Techniques: Activation Assay, Membrane, Binding Assay, Incubation, Flow Cytometry

ST6GAL1 catalyzes the α2,6-sialylation of LPAR4, EGFR, integrin α5, and integrin β1 in MDA-MB-231 cells. A, the cell lysates from Con-, ST6GAL1-KO-, ST6GAL1-Res- MDA-MB-231 cells were immunoprecipitated by SSA-agaroses and then blotted with antibodies against LPAR4, EGFR, integrin β1, and integrin α5. The whole-cell lysates were also subjected to WB with indicated antibodies. B, the cell lysates from Con- and ST3GAL4-OE- MDA-MB-231 cells were immunoprecipitated by SSA- and MAM-agaroses and then blotted with antibodies against integrin β1, EGFR, and integrin α5 separately. The whole-cell lysates were also subjected to WB with indicated antibodies. The relative ratios (the α2,6-sialylated LPAR4, EGFR, integrin α5, or integrin β1 versus total LPAR4, EGFR, integrin α5, or integrin β1, respectively) in ( A ) and (the α2,3-sialylated or α2,6-sialylated integrin β1, EGFR, or integrin α5 versus total integrin β1, EGFR, or integrin α5, respectively) in ( B ) are shown as the mean ± SD ( n = 3 biological replicates, ∗∗, p < 0.01; ∗∗∗, p < 0.001; and ∗∗∗∗, p < 0.0001 are determined by one-way ANOVA with Tukey's post hoc test and two-tail unpaired t test, respectively). WB, Western blot; ST6GAL1, β-galactoside α2,6-sialyltransferase 1; SSA, Sambucus sieboldiana agglutinin; Con, control; Res, rescue; EGFR, epidermal growth factor receptor; Maackia amurensis.

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: ST6GAL1 catalyzes the α2,6-sialylation of LPAR4, EGFR, integrin α5, and integrin β1 in MDA-MB-231 cells. A, the cell lysates from Con-, ST6GAL1-KO-, ST6GAL1-Res- MDA-MB-231 cells were immunoprecipitated by SSA-agaroses and then blotted with antibodies against LPAR4, EGFR, integrin β1, and integrin α5. The whole-cell lysates were also subjected to WB with indicated antibodies. B, the cell lysates from Con- and ST3GAL4-OE- MDA-MB-231 cells were immunoprecipitated by SSA- and MAM-agaroses and then blotted with antibodies against integrin β1, EGFR, and integrin α5 separately. The whole-cell lysates were also subjected to WB with indicated antibodies. The relative ratios (the α2,6-sialylated LPAR4, EGFR, integrin α5, or integrin β1 versus total LPAR4, EGFR, integrin α5, or integrin β1, respectively) in ( A ) and (the α2,3-sialylated or α2,6-sialylated integrin β1, EGFR, or integrin α5 versus total integrin β1, EGFR, or integrin α5, respectively) in ( B ) are shown as the mean ± SD ( n = 3 biological replicates, ∗∗, p < 0.01; ∗∗∗, p < 0.001; and ∗∗∗∗, p < 0.0001 are determined by one-way ANOVA with Tukey's post hoc test and two-tail unpaired t test, respectively). WB, Western blot; ST6GAL1, β-galactoside α2,6-sialyltransferase 1; SSA, Sambucus sieboldiana agglutinin; Con, control; Res, rescue; EGFR, epidermal growth factor receptor; Maackia amurensis.

Techniques: Immunoprecipitation, Western Blot, Control

ST6GAL1 mediates integrin β1–LPAR4/EGFR complex formation. A and B, the cell lysates from Con-, ST6GAL1-KO-, ST6GAL1-Res- MDA-MB-231 ( A ) and BT549 ( B ) cells were immunoprecipitated by anti-integrin β1 antibody and then blotted with antibodies against LPAR4, EGFR, and integrin β1. The whole-cell lysates were also subjected to WB with indicated antibodies. The relative ratios (the association of EGFR or LPAR4 with integrin β1, respectively) in ( A ) and ( B ) are presented as the mean ± SD ( n = 3 biological replicates, ∗∗∗∗, p < 0.0001 is determined by one-way ANOVA with Tukey's post hoc test). ST6GAL1, β-galactoside α2,6-sialyltransferase 1; Con, control; Res, rescue; EGFR, epidermal growth factor receptor; WB, Western blot.

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: ST6GAL1 mediates integrin β1–LPAR4/EGFR complex formation. A and B, the cell lysates from Con-, ST6GAL1-KO-, ST6GAL1-Res- MDA-MB-231 ( A ) and BT549 ( B ) cells were immunoprecipitated by anti-integrin β1 antibody and then blotted with antibodies against LPAR4, EGFR, and integrin β1. The whole-cell lysates were also subjected to WB with indicated antibodies. The relative ratios (the association of EGFR or LPAR4 with integrin β1, respectively) in ( A ) and ( B ) are presented as the mean ± SD ( n = 3 biological replicates, ∗∗∗∗, p < 0.0001 is determined by one-way ANOVA with Tukey's post hoc test). ST6GAL1, β-galactoside α2,6-sialyltransferase 1; Con, control; Res, rescue; EGFR, epidermal growth factor receptor; WB, Western blot.

Techniques: Immunoprecipitation, Control, Western Blot

Schematic diagram of the proposed molecular mechanism for negative regulation of Hippo signaling via ST6GAL1. Various upstream cell membrane receptors of the Hippo pathway have been identified, including the RTKs ( e.g. , EGFR), GPCRs ( e.g. , LPAR4), and integrins ( e.g. , integrin α5β1). The RTK, GPCR, and integrin signals transduced by growth factors (GFs, e.g. , EGF), extracellular factors ( e.g. , LPA), and the extracellular matrix (ECM, e.g. , FN) can facilitate Hippo pathway effectors ( e.g. , PI3K and FAK) association, which promote LATS1/2-mediated regulation of YAP. In the cells with ST6GAL1 expression ( left ), the cell membrane receptors, such as EGFR, LPAR4, and integrin α5β1, are modified by α2,6-sialylation, which mediate the integrin β1–EGFR/LPAR4 complex formation and in turn facilitate their responses to EGF, LPA, and FN, respectively. These signalings inactivate LATS1/2 kinases or induce the dephosphorylation of YAP, finally leading to hypophosphorylated YAP (p-YAP S127). Hypophosphorylated YAP accumulates in the nucleus, where it can bind to various transcription factors (TFs, e.g. , TEAD family) to enhance the expression of target genes ( e.g. , ANKRD1 , CTGF , and CYR61 ) expression that promote cell adhesion, spreading, proliferation, migration, and metastasis. The Hippo signaling can be inhibited by the verteporfin (VP) inhibitor, which targets YAP-TEAD activity. In the ST6GAL1 deficiency cells ( right ), the N -glycans on cell membrane receptors are without α2,6-sialylation, which exhibit weak integrin β1–EGFR/LPAR4 complex formation and delayed responses to EGF, LPA, and FN stimulation and activate the LATS1/2 kinases and phosphorylate YAP on S127. The phosphorylated YAP (p-YAP S127) is retained in the cytoplasm, inhibiting YAP/TEAD-dependent transcription. The p of the red background represents the activation of related proteins, while gray background represents the inactivation. LATS, large tumor suppressor kinase; YAP, yes-associated protein; ST6GAL1, β-galactoside α2,6-sialyltransferase 1; RTK, receptor tyrosine kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FN, fibronectin; GPCR, G protein–coupled receptor; GT, glycosyltransferase; LPA, lysophosphatidic acid; FAK, focal adhesion kinase.

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: Schematic diagram of the proposed molecular mechanism for negative regulation of Hippo signaling via ST6GAL1. Various upstream cell membrane receptors of the Hippo pathway have been identified, including the RTKs ( e.g. , EGFR), GPCRs ( e.g. , LPAR4), and integrins ( e.g. , integrin α5β1). The RTK, GPCR, and integrin signals transduced by growth factors (GFs, e.g. , EGF), extracellular factors ( e.g. , LPA), and the extracellular matrix (ECM, e.g. , FN) can facilitate Hippo pathway effectors ( e.g. , PI3K and FAK) association, which promote LATS1/2-mediated regulation of YAP. In the cells with ST6GAL1 expression ( left ), the cell membrane receptors, such as EGFR, LPAR4, and integrin α5β1, are modified by α2,6-sialylation, which mediate the integrin β1–EGFR/LPAR4 complex formation and in turn facilitate their responses to EGF, LPA, and FN, respectively. These signalings inactivate LATS1/2 kinases or induce the dephosphorylation of YAP, finally leading to hypophosphorylated YAP (p-YAP S127). Hypophosphorylated YAP accumulates in the nucleus, where it can bind to various transcription factors (TFs, e.g. , TEAD family) to enhance the expression of target genes ( e.g. , ANKRD1 , CTGF , and CYR61 ) expression that promote cell adhesion, spreading, proliferation, migration, and metastasis. The Hippo signaling can be inhibited by the verteporfin (VP) inhibitor, which targets YAP-TEAD activity. In the ST6GAL1 deficiency cells ( right ), the N -glycans on cell membrane receptors are without α2,6-sialylation, which exhibit weak integrin β1–EGFR/LPAR4 complex formation and delayed responses to EGF, LPA, and FN stimulation and activate the LATS1/2 kinases and phosphorylate YAP on S127. The phosphorylated YAP (p-YAP S127) is retained in the cytoplasm, inhibiting YAP/TEAD-dependent transcription. The p of the red background represents the activation of related proteins, while gray background represents the inactivation. LATS, large tumor suppressor kinase; YAP, yes-associated protein; ST6GAL1, β-galactoside α2,6-sialyltransferase 1; RTK, receptor tyrosine kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FN, fibronectin; GPCR, G protein–coupled receptor; GT, glycosyltransferase; LPA, lysophosphatidic acid; FAK, focal adhesion kinase.

Techniques: Membrane, Expressing, Modification, De-Phosphorylation Assay, Migration, Activity Assay, Activation Assay

Increased integrin β1 activity, elevated cell adhesion, and migration defects of ppm1f-/- MEFs are reverted by re-expression of wildtype PPM1F. A PPM1F-/- MEFs were transduced with lentiviral particles encoding human wildtype PPM1F (hWT) or human PPM1F D360 A (hDA) in a bi-cistronic expression cassette with GFP. In addition, PPM1F-/- MEFs and PPM1F +/+ cells were transduced with a lentivirus encoding GFP alone. WCLs of sorted cells were analyzed by Western blotting with the indicated antibodies; as controls, WCLs of 293 T cells transfected with the empty vector (mock), GFP (GFP) or murine PPM1F (mWT) were loaded. B MEFs as in ( A ) were seeded onto 1 µg/ml FN III9-12 for 2 h. Samples were fixed and stained for talin (upper panel) or the active integrin β1 (lower panel) before analysis by confocal microscopy; scale bar: 20 µm. Insets show higher magnification of boxed areas; scale bar: 5 µm. Arrowheads point to active integrin β1 or talin enrichment. C MEFs as in ( A ) were kept in suspension for 45 min and incubated for 15 min with 10 µg/ml FN III9-12 (FN). Samples were stained for total (Hmb1-1) or active β1 integrin (9EG7) and analyzed by flow cytometry, ≥ 10 000 counts. The mean fluorescence intensity (MFI) ratio of active to total β1 integrin was calculated and normalized to the wildtype sample (= 1). Scatter blots represent mean ± SEM of 4 independent experiments; statistics was performed using one-way ANOVA and Bonferroni post-hoc test ( p *** < 0.001, ns = not significant). D MEFs were seeded in triplicates onto fibronectin-coated wells for 60 min and cell adhesion was quantified. Representative pictures from cells seeded on 10 µg/ml FN (left panel); scale bar: 150 µm. Scatter blots represent mean ± SEM of 5 independent experiments performed in technical triplicates each. Values were normalized to MEF wildtype cells (= 1). Statistics was performed using one-way ANOVA, followed by Bonferroni post-hoc test (** p < 0.01, * p < 0.05, ns = not significant). E MEFs were seeded onto indicated fibronectin concentrations for 45 min, fixed and stained with DAPI and Phalloidin-Cy5. Samples were imaged using confocal microscopy. Representative images from cells seeded onto 10 µg/ml FN are shown; scale bar: 10 µm (left panel). Quantification of cell spreading. Boxes and whiskers indicate median with 95% confidence intervals from 2 independent experiments; n ≥ 90 cells. Statistics was performed using one-way ANOVA, followed by Bonferroni post-hoc test (*** p < 0.001, ns = not significant) (right panel). F Serum starved MEFs were stimulated by addition of 10% FCS and cell migration was monitored every 30 min for 12 h using time-lapse microscopy. Cell tracks were evaluated for velocity, covered distance and directionality. Boxes and whiskers indicate median with 95% confidence intervals from 2 independent experiments ( n = 30); Statistics was performed as in ( E ); *** p < 0.001, * p < 0.05, ns = not significant. See also Additional_File2

Journal: BMC Biology

Article Title: The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

doi: 10.1186/s12915-025-02254-3

Figure Lengend Snippet: Increased integrin β1 activity, elevated cell adhesion, and migration defects of ppm1f-/- MEFs are reverted by re-expression of wildtype PPM1F. A PPM1F-/- MEFs were transduced with lentiviral particles encoding human wildtype PPM1F (hWT) or human PPM1F D360 A (hDA) in a bi-cistronic expression cassette with GFP. In addition, PPM1F-/- MEFs and PPM1F +/+ cells were transduced with a lentivirus encoding GFP alone. WCLs of sorted cells were analyzed by Western blotting with the indicated antibodies; as controls, WCLs of 293 T cells transfected with the empty vector (mock), GFP (GFP) or murine PPM1F (mWT) were loaded. B MEFs as in ( A ) were seeded onto 1 µg/ml FN III9-12 for 2 h. Samples were fixed and stained for talin (upper panel) or the active integrin β1 (lower panel) before analysis by confocal microscopy; scale bar: 20 µm. Insets show higher magnification of boxed areas; scale bar: 5 µm. Arrowheads point to active integrin β1 or talin enrichment. C MEFs as in ( A ) were kept in suspension for 45 min and incubated for 15 min with 10 µg/ml FN III9-12 (FN). Samples were stained for total (Hmb1-1) or active β1 integrin (9EG7) and analyzed by flow cytometry, ≥ 10 000 counts. The mean fluorescence intensity (MFI) ratio of active to total β1 integrin was calculated and normalized to the wildtype sample (= 1). Scatter blots represent mean ± SEM of 4 independent experiments; statistics was performed using one-way ANOVA and Bonferroni post-hoc test ( p *** < 0.001, ns = not significant). D MEFs were seeded in triplicates onto fibronectin-coated wells for 60 min and cell adhesion was quantified. Representative pictures from cells seeded on 10 µg/ml FN (left panel); scale bar: 150 µm. Scatter blots represent mean ± SEM of 5 independent experiments performed in technical triplicates each. Values were normalized to MEF wildtype cells (= 1). Statistics was performed using one-way ANOVA, followed by Bonferroni post-hoc test (** p < 0.01, * p < 0.05, ns = not significant). E MEFs were seeded onto indicated fibronectin concentrations for 45 min, fixed and stained with DAPI and Phalloidin-Cy5. Samples were imaged using confocal microscopy. Representative images from cells seeded onto 10 µg/ml FN are shown; scale bar: 10 µm (left panel). Quantification of cell spreading. Boxes and whiskers indicate median with 95% confidence intervals from 2 independent experiments; n ≥ 90 cells. Statistics was performed using one-way ANOVA, followed by Bonferroni post-hoc test (*** p < 0.001, ns = not significant) (right panel). F Serum starved MEFs were stimulated by addition of 10% FCS and cell migration was monitored every 30 min for 12 h using time-lapse microscopy. Cell tracks were evaluated for velocity, covered distance and directionality. Boxes and whiskers indicate median with 95% confidence intervals from 2 independent experiments ( n = 30); Statistics was performed as in ( E ); *** p < 0.001, * p < 0.05, ns = not significant. See also Additional_File2

Article Snippet: The following antibodies were used with the corresponding dilutions for western blot analysis (WB), immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation (IP), or integrin activity assay (IA): α-Actinin (BM75.2, mouse anti-human, Abcam; 1:1000 WB), α 1 -integrin (TS2/7, mouse anti-human/anti-mouse, Abcam; 1:50 IF), α 2 -integrin (6 F1, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 3 -integrin (P1B5, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 5 -integrin (BIIG2, rat anti-human/anti-mouse, DSHB; 1:10 IF), α v -integrin (PE-P2 W7 mouse anti-human/anti-mouse, sc-9969; IF 1:300), β 1 -integrin (HMβ1-1, armenian hamster anti-mouse, Bio Legend; 1:300 IF; AIIB2, rat anti-human/anti-mouse, DSHB; 1:600 IF, IA; M-106, rabbit anti-mouse/anti-human, Santa Cruz; 1:500 WB; D2E5, rabbit anti-human, Cell Signaling; 1:1000 WB), human β 1 -integrin (P5D2, mouse anti-human, DSHB, 2.5 μg IP; 9EG7, rat anti- human, DSHB 2.5 μg IP; AIIB2, rat anti-human, DSHB; 2.5 μg IP), β 3 -integrin (2 C9.G3, arm. hamster anti-mouse, eBioscience; 1:300 IF; PM6/13, mouse anti-human, Abcam; 1:100 IF), β 5 -integrin (KN-52, mouse anti-mouse/human, eBioscience; IF 1:300), Focal adhesion kinase (FAK) (77, mouse anti-human, BD; 1:250 WB), integrin-linked kinase (ILK) (EP1593Y, rabbit anti-human, Epitomics; 1:800 WB), Kindlin-2 (3 A3, mouse anti-human, Millipore; 1:200 WB, 1:250 IF), Laminin (ab11575, rabbit anti-mouse, Abcam; 1:300 IHC), Nestin (rat-401, anti-mouse, Millipore; IHC 1:200), Paxillin (5H11, mouse monoclonal, Thermo Scientific; 1:1000 WB), hPPM1F (17,020–1-AP, rabbit anti-human, Protein-Tech; 1:1000 WB), mPPM1F (#1147, rabbit anti-mouse PPM1F; generated at the central animal care facility; University of Konstanz; 1:200 WB; see Additional File2: Fig. S2), FilaminA (EP2405Y, IgG, rabbit anti-human, Epitomics; 1:125.000 WB), Tubulin (E7, IgG1, mouse anti-human, DSHB; 1:1000), Talin (8 d4, mouse anti-human, Thermo Scientific; 1:800 WB, 1:40 IF), Vinculin (hVIN-1, mouse anti-human, Sigma; 1:2000 WB, 1:200 IF), Zyxin (Zol301, mouse anti-human, Abcam; 1:1000 WB), Dylight488-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy3-conjugated goat anti-rabbit IgG (Jackson; 1:200), Cy3-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy5-conjugated goat anti-mouse IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-rat IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-Armenian Hamster IgG (Jackson; 1:200), HRP-conjugated goat anti-mouse IgG (Jackson; WB 1:10 000), HRP-conjugated goat anti-rat IgG (Santa Cruz; 1:250), HRP-conjugated goat anti-rabbit IgG (Jackson; WB 1:3000), unspecific control IgG (anti-mouse, 96/1, generated at the Tierforschungsanlage; University of Konstanz; anti-rat, MJ7/18 Endoglin, DSHB).

Techniques: Activity Assay, Migration, Expressing, Transduction, Western Blot, Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Suspension, Incubation, Flow Cytometry, Fluorescence, Time-lapse Microscopy

PPM1F contributes to the invasive phenotype of tumor cells. A WCLs from indicated cancer cell lines were analyzed by Western blotting with α-human PPM1F or α-integrin β1 antibodies. α-Tubulin antibody was used as loading control. B , C Indicated serum-starved cancer cells were seeded on top of a Matrigel basement membrane (30 µg/100 µl) in Boyden chamber cell invasion assays using 20% FCS as stimulus or 2% BSA to evaluate random invasion activity. NIH3 T3 cells served as non-invasive control cells. Representative pictures of the lower porous membrane surface (20x) are shown in (B); scale bar: 50 µm. Crystal violet-stained cells can be distinguished from the 8 µm membrane pores. Cells were evaluated for invasion after 24 h by dye elution with 10% acetic acid and absorbance measurement at 590 nm. Graph in ( C ) shows quantified means ± SEM from three independent experiments. Statistics was performed using one-way ANOVA and Bonferroni post-hoc test ( p *** < 0.001, p ** < 0.01, ns = not significant). D MCF-7 cells were stably transduced with lentiviral particles harboring a bicistronic GFP and hPPM1F wildtype or hPPM1F D360 A expression cassette and single-cell sorted via flow cytometry for GFP positive cells to obtain a mixed population of PPM1F-overexpressing MCF-7 cells (PPM1F + + and PPM1F D360 A + +). WCL of the wildtype and modified cell lines were analyzed by Western blotting with indicated antibodies. α-tubulin antibody (lowest panel) served as loading control. E Serum-starved cells from ( D ) were seeded on top of a Matrigel base (30 µg/100 µl) in Boyden chambers. Cell invasion was stimulated by addition of 20% FCS or 2% BSA to the lower chamber. Representative pictures of the lower porous membrane surface (20x) are shown; scale bar: 50 µm. Crystal violet-stained cells can be distinguished from the 8 µm membrane pores. Invasion was quantified by dye elution. Graph (right) shows means ± SEM from four independent experiments performed in triplicate. Statistics as in ( C )

Journal: BMC Biology

Article Title: The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

doi: 10.1186/s12915-025-02254-3

Figure Lengend Snippet: PPM1F contributes to the invasive phenotype of tumor cells. A WCLs from indicated cancer cell lines were analyzed by Western blotting with α-human PPM1F or α-integrin β1 antibodies. α-Tubulin antibody was used as loading control. B , C Indicated serum-starved cancer cells were seeded on top of a Matrigel basement membrane (30 µg/100 µl) in Boyden chamber cell invasion assays using 20% FCS as stimulus or 2% BSA to evaluate random invasion activity. NIH3 T3 cells served as non-invasive control cells. Representative pictures of the lower porous membrane surface (20x) are shown in (B); scale bar: 50 µm. Crystal violet-stained cells can be distinguished from the 8 µm membrane pores. Cells were evaluated for invasion after 24 h by dye elution with 10% acetic acid and absorbance measurement at 590 nm. Graph in ( C ) shows quantified means ± SEM from three independent experiments. Statistics was performed using one-way ANOVA and Bonferroni post-hoc test ( p *** < 0.001, p ** < 0.01, ns = not significant). D MCF-7 cells were stably transduced with lentiviral particles harboring a bicistronic GFP and hPPM1F wildtype or hPPM1F D360 A expression cassette and single-cell sorted via flow cytometry for GFP positive cells to obtain a mixed population of PPM1F-overexpressing MCF-7 cells (PPM1F + + and PPM1F D360 A + +). WCL of the wildtype and modified cell lines were analyzed by Western blotting with indicated antibodies. α-tubulin antibody (lowest panel) served as loading control. E Serum-starved cells from ( D ) were seeded on top of a Matrigel base (30 µg/100 µl) in Boyden chambers. Cell invasion was stimulated by addition of 20% FCS or 2% BSA to the lower chamber. Representative pictures of the lower porous membrane surface (20x) are shown; scale bar: 50 µm. Crystal violet-stained cells can be distinguished from the 8 µm membrane pores. Invasion was quantified by dye elution. Graph (right) shows means ± SEM from four independent experiments performed in triplicate. Statistics as in ( C )

Techniques: Western Blot, Control, Membrane, Activity Assay, Staining, Stable Transfection, Transduction, Expressing, Flow Cytometry, Modification

Genetic deletion of PPM1F in tumor cells diminishes matrix invasion and integrin phosphorylation. A WCLs from A172 wildtype cells and two clonal PPM1F KO cell lines (1 and 2) were analyzed by Western blotting using the indicated antibodies. α-Tubulin antibody was used as loading control. B Serum starved A172 wildtype cells and PPM1F KO cell lines (clone 1 and clone 2) were seeded in triplicate onto fibronectin-, vitronectin-, or 2% BSA-coated wells for 60 min either in presence of 50 µM cilengitide or DMSO as control. Wells were washed and adherent cells were stained with crystal violet. Representative pictures are shown; scale bar: 150 µm. C Adherent crystal violett stained cells from ( B ) were quantified by dye elution. Graph depicts individual values as well as mean ± SEM of 4 independent experiments performed in technical triplicates. Statistics was performed using one-way ANOVA, followed by Bonferroni post-hoc test (*** p < 0.001; ** p < 0.01; p * < 0.05; ns = not significant) and shown for the PPM1F knock-out clones in relation to the A172 wildtype cells. D Serum-starved cells as in ( C ) were seeded on top of a Matrigel base (30 µg/100 µl) in Boyden chambers and cell invasion was stimulated by addition of 20% FCS or 2% BSA to the lower chamber. Cells were evaluated for invasion after 24 h and representative pictures of the lower porous membrane surface (20x) are shown; scale bar: 50 µm. Crystal violet-stained cells can be distinguished from the 8 µm membrane pores (left). Invasion assays were quantified by dye elution. Graph depicts individual values as well as means ± SEM from four independent experiments performed in triplicate. Statistics as in ( C ). See also Additional_File4 and Additional_File5

Journal: BMC Biology

Article Title: The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

doi: 10.1186/s12915-025-02254-3

Figure Lengend Snippet: Genetic deletion of PPM1F in tumor cells diminishes matrix invasion and integrin phosphorylation. A WCLs from A172 wildtype cells and two clonal PPM1F KO cell lines (1 and 2) were analyzed by Western blotting using the indicated antibodies. α-Tubulin antibody was used as loading control. B Serum starved A172 wildtype cells and PPM1F KO cell lines (clone 1 and clone 2) were seeded in triplicate onto fibronectin-, vitronectin-, or 2% BSA-coated wells for 60 min either in presence of 50 µM cilengitide or DMSO as control. Wells were washed and adherent cells were stained with crystal violet. Representative pictures are shown; scale bar: 150 µm. C Adherent crystal violett stained cells from ( B ) were quantified by dye elution. Graph depicts individual values as well as mean ± SEM of 4 independent experiments performed in technical triplicates. Statistics was performed using one-way ANOVA, followed by Bonferroni post-hoc test (*** p < 0.001; ** p < 0.01; p * < 0.05; ns = not significant) and shown for the PPM1F knock-out clones in relation to the A172 wildtype cells. D Serum-starved cells as in ( C ) were seeded on top of a Matrigel base (30 µg/100 µl) in Boyden chambers and cell invasion was stimulated by addition of 20% FCS or 2% BSA to the lower chamber. Cells were evaluated for invasion after 24 h and representative pictures of the lower porous membrane surface (20x) are shown; scale bar: 50 µm. Crystal violet-stained cells can be distinguished from the 8 µm membrane pores (left). Invasion assays were quantified by dye elution. Graph depicts individual values as well as means ± SEM from four independent experiments performed in triplicate. Statistics as in ( C ). See also Additional_File4 and Additional_File5

Techniques: Phospho-proteomics, Western Blot, Control, Staining, Knock-Out, Clone Assay, Membrane

Increased integrin-based cell adhesion in PPM1F-deficient cells prohibits cell spreading despite elevated PAK activity. A Serum-starved A172 wildtype, sgRNA control and PPM1F KO cells were seeded onto 2 µg/ml FN III9-12 for 45 min and WCLs were subjected to Western blotting with indicated antibodies (left panel). Graphs (right panel) show densitometric quantification of band intensities from pThr402PAK2 versus PAK antibody signal for the indicated samples from 5 independent experiments; wildtype was set to 1. Statistics were performed using one-way ANOVA, followed by Bonferroni post-hoc test (* p < 0.05, ns = not significant). B Serum-starved A172 wildtype and PPM1F KO cells were seeded onto 2 µg/ml FN III9-12 for 1.5 h, fixed and F-actin was stained. Samples were imaged using confocal microscopy. Representative pictures are shown; scale bar: 20 µm. C Cells as in ( B ) were seeded for 2 h on surfaces coated with 10 µg/ml fibronectin or poly-L-lysine, before fixation, F-actin staining and analysis by confocal microscopy; scale bar: 10 µm. D Spreading assays were performed with serum-starved A172 wildtype and PPM1F KO cells re-expressing mKate2 or re-expressing PPM1F-mKate2 cells, pre-treated with 5 µM DMSO or FRAX597 (PAK1-3 inhibitor) for 45 min in suspension before seeding onto 2 µg/ml FN III9-12 for 1.5 h. Cells were fixed, stained for F-actin and the covered area was quantified in ImageJ. Boxes and whiskers indicate median with 95% confidence intervals from two independent experiments; n ≥ 30 cells; dots indicate outliers. Statistics was performed using one-way ANOVA, followed by post-hoc Bonferroni test, (*** p < 0.001, ns = not significant). E Serum-starved cells as in ( D ) were pre-treated with 5 µM DMSO or FRAX597 (PAK1-3 inhibitor) for 45 min in suspension before seeded onto 2 µg/ml FN III9-12 for 1.5 h. Cells were fixed and stained for active integrin β1. Cells were imaged by confocal microscopy. Representative pictures are shown; scale bar: 10 µm. See also Additional_File6 and Additional_File7

Journal: BMC Biology

Article Title: The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

doi: 10.1186/s12915-025-02254-3

Figure Lengend Snippet: Increased integrin-based cell adhesion in PPM1F-deficient cells prohibits cell spreading despite elevated PAK activity. A Serum-starved A172 wildtype, sgRNA control and PPM1F KO cells were seeded onto 2 µg/ml FN III9-12 for 45 min and WCLs were subjected to Western blotting with indicated antibodies (left panel). Graphs (right panel) show densitometric quantification of band intensities from pThr402PAK2 versus PAK antibody signal for the indicated samples from 5 independent experiments; wildtype was set to 1. Statistics were performed using one-way ANOVA, followed by Bonferroni post-hoc test (* p < 0.05, ns = not significant). B Serum-starved A172 wildtype and PPM1F KO cells were seeded onto 2 µg/ml FN III9-12 for 1.5 h, fixed and F-actin was stained. Samples were imaged using confocal microscopy. Representative pictures are shown; scale bar: 20 µm. C Cells as in ( B ) were seeded for 2 h on surfaces coated with 10 µg/ml fibronectin or poly-L-lysine, before fixation, F-actin staining and analysis by confocal microscopy; scale bar: 10 µm. D Spreading assays were performed with serum-starved A172 wildtype and PPM1F KO cells re-expressing mKate2 or re-expressing PPM1F-mKate2 cells, pre-treated with 5 µM DMSO or FRAX597 (PAK1-3 inhibitor) for 45 min in suspension before seeding onto 2 µg/ml FN III9-12 for 1.5 h. Cells were fixed, stained for F-actin and the covered area was quantified in ImageJ. Boxes and whiskers indicate median with 95% confidence intervals from two independent experiments; n ≥ 30 cells; dots indicate outliers. Statistics was performed using one-way ANOVA, followed by post-hoc Bonferroni test, (*** p < 0.001, ns = not significant). E Serum-starved cells as in ( D ) were pre-treated with 5 µM DMSO or FRAX597 (PAK1-3 inhibitor) for 45 min in suspension before seeded onto 2 µg/ml FN III9-12 for 1.5 h. Cells were fixed and stained for active integrin β1. Cells were imaged by confocal microscopy. Representative pictures are shown; scale bar: 10 µm. See also Additional_File6 and Additional_File7

Techniques: Activity Assay, Control, Western Blot, Staining, Confocal Microscopy, Expressing, Suspension

Review

human β1 integrin (Developmental Studies Hybridoma Bank)

Structured Review

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