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: Briefly, fresh sheep red blood cells (RBCs) (Thermo Scientific) labelled with 2 μM DDAO-AM (Invitrogen) were incubated with sub-agglutinating concentrations of polyclonal rabbit IgM anti-sheep RBC cells (MyBioSource) and later treated with 10% C5-deficient human serum (Sigma) for complement opsonization.

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