Journal: STAR Protocols

Article Title: Protocol for reconstituting peptides/peptidomimetics from DMSO to aqueous buffers for circular dichroism analyses

doi: 10.1016/j.xpro.2024.102850

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Article Snippet: S-TBS: a stapled peptide derived from the Talin-binding site in RIAM (sequence: NEDIDQMFS TLLGE(S5)Dll(S5)TQS) , New England Peptide , Lot# LB7720.

Techniques: Recombinant, Sequencing, Purification, Derivative Assay, Software

Journal: Frontiers in Immunology

Article Title: Expression of the phagocytic receptors α M β 2 and α X β 2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells

doi: 10.3389/fimmu.2022.951280

Figure Lengend Snippet: Knockout of either RIAM, VASP or Vinculin, abolishes phagocytosis. (A, B) Newly generated HL-60 Vinculin (Vcl), RIAM and VASP knockout monoclonal cell lines were tested for integrin related protein expression by western blot. (C) Quantification of protein expression in neutrophil-like HL-60 cells and derived knockouts analyzed by western blot. Results are represented as relative to HL-60 levels and are from 5 independent experiments. (D, E) Phagocytic cells were challenged with complement opsonized sheep red blood cells (C3-RBC) after being stimulated with 1 mM MnCl 2 or left unstimulated, and Association (AI) and Phagocytic (PI) indexes were obtained. Data are normalized with respect to the AI of unstimulated C3-RBC-challenged HL-60 cells (control cells). Data are presented as mean ± SD, where the error bars denote standard deviation. Significance (ANOVA) has been calculated with respect to HL-60 controls, ** denotes p<0.01 and ****,p<0.0001.

Article Snippet: 50 μg protein were separated by SDS-PAGE electrotransferred to a nitrocellulose membrane, blocked and incubated with the following anti-human primary antibodies: rabbit IgG anti-VASP, (Cell Signaling), mouse IgG anti-α-Tubulin, (Sigma), mouse IgG anti-Vinculin (H-10 clone, Santa Cruz), sheep IgG anti-RIAM (R&D Systems) and mouse IgG anti-Talin (8D4 clone, Sigma).

Techniques: Knock-Out, Generated, Expressing, Western Blot, Derivative Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Expression of the phagocytic receptors α M β 2 and α X β 2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells

doi: 10.3389/fimmu.2022.951280

Figure Lengend Snippet: α M and α X expression is dependent on Vinculin, RIAM and VASP expression. (A) Vinculin (Vcl), RIAM and VASP knockout cell lines and HL-60 parental cells were differentiated into neutrophilic-like cells with 1 μM all- trans retinoic acid (RA) and stained with monoclonal antibodies specific for α M , α L, α X, β 1 , β 2 integrin subunits. The geometric mean fluorescence intensity (GMFI) was obtained by flow cytometry and data represented as relative to HL-60 levels and are from 24 independent experiments done in duplicate. (B) Expression of ITGAM (α M ), ITGAX (α X ), and ITGB2 (β 2 ) mRNA levels was determined by RT-qPCR in neutrophil-like RIAM, VASP and Vcl HL-60 knockouts. Results are represented as relative to GAPDH mRNA and are from 3 independent experiments done in triplicate. Data are presented as mean ± SD, where the error bars denote standard deviation. Significance (ANOVA) has been calculated with respect to HL-60 controls, *** denotes p<0.005, and ****, p<0.0001.

Article Snippet: 50 μg protein were separated by SDS-PAGE electrotransferred to a nitrocellulose membrane, blocked and incubated with the following anti-human primary antibodies: rabbit IgG anti-VASP, (Cell Signaling), mouse IgG anti-α-Tubulin, (Sigma), mouse IgG anti-Vinculin (H-10 clone, Santa Cruz), sheep IgG anti-RIAM (R&D Systems) and mouse IgG anti-Talin (8D4 clone, Sigma).

Techniques: Expressing, Knock-Out, Staining, Fluorescence, Flow Cytometry, Quantitative RT-PCR, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Expression of the phagocytic receptors α M β 2 and α X β 2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells

doi: 10.3389/fimmu.2022.951280

Figure Lengend Snippet: Upregulation of α M and α X expression during HL-60 neutrophilic differentiation depends on Vinculin, RIAM and VASP expression and is related cellular F- actin content. (A, B) Vinculin (Vcl), RIAM and VASP knockout cell lines and HL-60 parental cells were differentiated into neutrophilic-like cells with 1 μM all- trans retinoic acid treatment (RA+) or maintained undifferentiated (RA-), and expression of α M and α X integrins was analyzed by flow cytometry. (C) Cellular F-actin content was analyzed using fluorescently labeled phalloidin in HL-60 knockout cell lines and parental cells, in undifferentiated or differentiated cells. (D, E) Vinculin, RIAM and VASP knockout cell lines and HL-60 parental cells were treated with a 2 h 1 µM jasplakinolide stimulation, followed by a 24 h resting period during neutrophilic differentiation. Then, expression of α M and α X integrins was analyzed by flow cytometry. (F) Expression of α M integrin was analyzed in VASP F6 and F10 knockout clones and in VASP knock-in polyclonal cell lines F6 KI and F10 KI. Results are represented as GMFI relative to HL-60 wild type levels and are from at least 3 independent experiments done in triplicate. Data are presented as mean ± SD, where the error bars denote standard deviation. Significance (ANOVA) has been calculated with respect to HL-60 controls, ** denotes p<0.01, *** p<0.005, and **** p<0.0001, and ns denotes no significance.

Article Snippet: 50 μg protein were separated by SDS-PAGE electrotransferred to a nitrocellulose membrane, blocked and incubated with the following anti-human primary antibodies: rabbit IgG anti-VASP, (Cell Signaling), mouse IgG anti-α-Tubulin, (Sigma), mouse IgG anti-Vinculin (H-10 clone, Santa Cruz), sheep IgG anti-RIAM (R&D Systems) and mouse IgG anti-Talin (8D4 clone, Sigma).

Techniques: Expressing, Knock-Out, Flow Cytometry, Labeling, Clone Assay, Knock-In, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Expression of the phagocytic receptors α M β 2 and α X β 2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells

doi: 10.3389/fimmu.2022.951280

Figure Lengend Snippet: MRTF-A distribution is altered in Vinculin, RIAM and VASP knockouts. (A) HL-60 parental cells and the knockouts for Vinculin (Vcl), RIAM and VASP were differentiated into neutrophil-like cells, fixed, permeabilized and fluorescently labelled with an anti-MRTF-A mAb, DAPI for nuclear staining and anti-β 1 integrin to delimit the plasma membrane. Images show representative results from 3 independent experiments analyzed by confocal fluorescence microscopy. Bars indicate 5 µm. (B) Quantification of MRFT-A nuclear distribution in these images is represented. Results are represented as relative to the wild-type nuclear ratio and are from 3 independent experiments with at least 50 cells. Data are presented as mean ± SD, where the error bars denote standard deviation. Significance (ANOVA) has been calculated with respect to HL-60 controls, **** denotes p<0.0001.

Article Snippet: 50 μg protein were separated by SDS-PAGE electrotransferred to a nitrocellulose membrane, blocked and incubated with the following anti-human primary antibodies: rabbit IgG anti-VASP, (Cell Signaling), mouse IgG anti-α-Tubulin, (Sigma), mouse IgG anti-Vinculin (H-10 clone, Santa Cruz), sheep IgG anti-RIAM (R&D Systems) and mouse IgG anti-Talin (8D4 clone, Sigma).

Techniques: Staining, Membrane, Fluorescence, Microscopy, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Expression of the phagocytic receptors α M β 2 and α X β 2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells

doi: 10.3389/fimmu.2022.951280

Figure Lengend Snippet: FHL-2 nuclear translocation is enhanced in Vinculin, RIAM and VASP knockouts. (A) HL-60 parental cells and the knockouts for Vcl, RIAM, VASP HL-60 cells were differentiated into neutrophilic-like cells with 1 μM all- trans retinoic acid for 48 h, fixed, permeabilized and fluorescently labelled using anti-FHL-2 and anti-β 1 integrin antibodies and DAPI. Confocal microscopy images were analyzed using the ImageJ software package and are representative results from 3 independent experiments. Bars indicate 5 µm. (B, C) Subcellular distribution of FHL-2. The graphs represent the quantification of images. Results are represented as relative to the wild-type nuclear ratio and are from 3 independent experiments with at least 50 cells. Data are presented as mean ± SD, where the error bars denote standard deviation. Significance (ANOVA) has been calculated with respect to HL-60 controls, ***denotes p<0.005, and ****, p<0.0001.

Article Snippet: 50 μg protein were separated by SDS-PAGE electrotransferred to a nitrocellulose membrane, blocked and incubated with the following anti-human primary antibodies: rabbit IgG anti-VASP, (Cell Signaling), mouse IgG anti-α-Tubulin, (Sigma), mouse IgG anti-Vinculin (H-10 clone, Santa Cruz), sheep IgG anti-RIAM (R&D Systems) and mouse IgG anti-Talin (8D4 clone, Sigma).

Techniques: Translocation Assay, Confocal Microscopy, Software, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Expression of the phagocytic receptors α M β 2 and α X β 2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells

doi: 10.3389/fimmu.2022.951280

Figure Lengend Snippet: Jasplakinolide induces MRTF-A nuclear translocation in Vinculin, RIAM and VASP knockouts. (A) Differentiated HL-60 parental cells and HL-60 knockouts for Vcl, RIAM and VASP were adhered to slides, then subjected to a 1 µM 2 h jasplakinolide stimulation, fixed, permeabilized and fluorescently stained to determine MRTF-A localization. β 1 integrin was used to delimit the plasma membrane and DAPI was used as a nuclear stain. Confocal microscopy images were analyzed using the ImageJ package and are representative results from 3 independent experiments. Bars indicate 5 µm. (B) Quantification images from the untreated cells shown on Figure 4 and jasplakinolide-treated cells on (A) . Results are represented as relative to the wild-type nuclear ratio and are from 3 independent experiments with at least 50 cells. Data are presented as mean ± SD, where the error bars denote standard deviation. Significance (ANOVA) has been calculated with respect to HL-60 controls, * denotes p<0.05, ***,p<0.005, and ****,p<0.0001.

Article Snippet: 50 μg protein were separated by SDS-PAGE electrotransferred to a nitrocellulose membrane, blocked and incubated with the following anti-human primary antibodies: rabbit IgG anti-VASP, (Cell Signaling), mouse IgG anti-α-Tubulin, (Sigma), mouse IgG anti-Vinculin (H-10 clone, Santa Cruz), sheep IgG anti-RIAM (R&D Systems) and mouse IgG anti-Talin (8D4 clone, Sigma).

Techniques: Translocation Assay, Staining, Membrane, Confocal Microscopy, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Expression of the phagocytic receptors α M β 2 and α X β 2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells

doi: 10.3389/fimmu.2022.951280

Figure Lengend Snippet: Jasplakinolide reduces nuclear localization of FHL-2 in Vinculin, RIAM and VASP knockouts. (A) Differentiated HL-60 parental cells and HL-60 knockouts for Vcl, RIAM and VASP were adhered to slides, then subjected to a 1 µ M 2 h jasplakinolide stimulation, fixed, permeabilized and fluorescently stained to determine FHL-2 localization. β 1 integrin was used to delimit the plasma membrane and DAPI was used as a nuclear stain. Confocal microscopy images were analyzed using the ImageJ package and are representative results from 3 independent experiments. Bars indicate 5 μm. (B, C) Subcellular distribution of FHL-2. The graphs represent the quantification of images from untreated cells shown on Figure 5 and jasplakinolide-treated cells shown on (A) . Results are represented as relative to the parental HL-60 membrane and nuclear ratio and are from 3 independent experiments with at least 50 cells. Data are presented as mean ± SD, where the error bars denote standard deviation. Significance (ANOVA) has been calculated with respect to HL-60 controls, ** denotes p<0.01, ***, p<0.005, and ****, p<0.0001.

Article Snippet: 50 μg protein were separated by SDS-PAGE electrotransferred to a nitrocellulose membrane, blocked and incubated with the following anti-human primary antibodies: rabbit IgG anti-VASP, (Cell Signaling), mouse IgG anti-α-Tubulin, (Sigma), mouse IgG anti-Vinculin (H-10 clone, Santa Cruz), sheep IgG anti-RIAM (R&D Systems) and mouse IgG anti-Talin (8D4 clone, Sigma).

Techniques: Staining, Membrane, Confocal Microscopy, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Expression of the phagocytic receptors α M β 2 and α X β 2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells

doi: 10.3389/fimmu.2022.951280

Figure Lengend Snippet: A model for integrin α M and α X upregulation dependent on RIAM, VASP and Vinculin expression. The expression of RIAM, VASP and Vinculin downstream of receptor signaling, contributes to increase the polymerization of actin. The reduced G-actin content allows MRTF-A translocation to the nucleus where, in combination with the SRF transcription factor promotes α M expression predominantly. Simultaneously, FHL-2 is sequestered in the plasma membrane, possibly by interacting with integrins or associated components, reducing FHL-2 co-repressor activity on SRF and allowing integrin expression. In addition, RIAM, VASP and Vinculin could regulate the activity of other potential transcription factors involved in the control α M and α X expression.

Article Snippet: 50 μg protein were separated by SDS-PAGE electrotransferred to a nitrocellulose membrane, blocked and incubated with the following anti-human primary antibodies: rabbit IgG anti-VASP, (Cell Signaling), mouse IgG anti-α-Tubulin, (Sigma), mouse IgG anti-Vinculin (H-10 clone, Santa Cruz), sheep IgG anti-RIAM (R&D Systems) and mouse IgG anti-Talin (8D4 clone, Sigma).

Techniques: Expressing, Translocation Assay, Membrane, Activity Assay