American Society of Hematology

Figure 3

$Figure 3. Role of PLD1 and PLCγ1 in FcγRI- and FcγRIIa-mediated signaling. (A) FcγRI- and FcγRIIa-mediated PKC activity measured as the phosphorylation rate (pmol/min) in whole-cell lysates. Antibody-mediated FcγR aggregation in untreated cells (XL FcγRI (antibody 10.1) or XL FcγRIIa (antibody 3D3), cells pretreated with the antisense oligos against PLD1 (XL FcγRI a.s.PLD1 or XL FcγRIIa a.s.PLD1), or antisense oligos against PLCγ1 (XL FcγRI a.s.PLCγ1 or XL FcγRIIa a.s.PLCγ1).(B) FcγRI-mediated PKC isoform translocation requires PLD1. Immunoblot analysis of the PKC isoenzymes translocated to the nuclear-free membrane fraction after antibody-mediated FcγRI aggregation in control cells (untreated) and cells pretreated with the antisenseoligos against PLD1 or PLCγ1 (a.s.PLD1 or a.s.PLCγ1). (C) FcγRIIa-mediated PKC isoform translocation requires PLCγ1. The same analyses applied to cells after FcγRIIa activation. (D) FcγRI and FcγRIIa-mediated degranulation. (i) β-Hexosaminidase release in resting cells (basal) or after antibody-mediated FcγRI aggregation in control cells (XL FcγRI) compared with cells pretreated with antisense oligonucleotides to either PLD1 (XL FcγRI a.s.PLD1) or PLCγ1 (XL FcγRI a.s.PLCγ1). (ii) The same analyses applied to cells after FcγRIIa activation. (E) FcγRI and FcγRIIa-mediated activation of NADPH oxidative burst. (i) Superoxide production in response to antibody-mediated FcγRI activation in control cells (XL FcγRI) compared with cells pretreated with antisense oligos to either PLD1 (XL FcγRI a.s.PLD1) or PLCγ1 (XL FcγRI a.s.PLCγ1). (ii) The same analyses applied to cells after FcγRIIa activation. Results expressed are the mean ± SD of triplicate samples of 3 independent experiments. (Fi) Primary human monocytes pretreated with anti-PLD1 or anti-PLCγ1 oligonucleotides down-regulate PLD1 and PLCγ1, respectively, compared with α-tubulin controls. (Fii) Monocytes treated with anti-PLD1 oligonucleotides are impaired in their cytosolic calcium signaling after activation of FcγRI by receptor cross-linking using antibody clone 10.1. (Fiii) Monocytes treated with anti-PLCγ1 oligonucleotides are impaired in their cytosolic calcium signaling after activation of FcγRIIa by receptor cross-linking using antibody clone 3D3.$

Role of PLD1 and PLCγ1 in FcγRI- and FcγRIIa-mediated signaling. (A) FcγRI- and FcγRIIa-mediated PKC activity measured as the phosphorylation rate (pmol/min) in whole-cell lysates. Antibody-mediated FcγR aggregation in untreated cells (XL FcγRI (antibody 10.1) or XL FcγRIIa (antibody 3D3), cells pretreated with the antisense oligos against PLD1 (XL FcγRI a.s.PLD1 or XL FcγRIIa a.s.PLD1), or antisense oligos against PLCγ1 (XL FcγRI a.s.PLCγ1 or XL FcγRIIa a.s.PLCγ1).(B) FcγRI-mediated PKC isoform translocation requires PLD1. Immunoblot analysis of the PKC isoenzymes translocated to the nuclear-free membrane fraction after antibody-mediated FcγRI aggregation in control cells (untreated) and cells pretreated with the antisenseoligos against PLD1 or PLCγ1 (a.s.PLD1 or a.s.PLCγ1). (C) FcγRIIa-mediated PKC isoform translocation requires PLCγ1. The same analyses applied to cells after FcγRIIa activation. (D) FcγRI and FcγRIIa-mediated degranulation. (i) β-Hexosaminidase release in resting cells (basal) or after antibody-mediated FcγRI aggregation in control cells (XL FcγRI) compared with cells pretreated with antisense oligonucleotides to either PLD1 (XL FcγRI a.s.PLD1) or PLCγ1 (XL FcγRI a.s.PLCγ1). (ii) The same analyses applied to cells after FcγRIIa activation. (E) FcγRI and FcγRIIa-mediated activation of NADPH oxidative burst. (i) Superoxide production in response to antibody-mediated FcγRI activation in control cells (XL FcγRI) compared with cells pretreated with antisense oligos to either PLD1 (XL FcγRI a.s.PLD1) or PLCγ1 (XL FcγRI a.s.PLCγ1). (ii) The same analyses applied to cells after FcγRIIa activation. Results expressed are the mean ± SD of triplicate samples of 3 independent experiments. (Fi) Primary human monocytes pretreated with anti-PLD1 or anti-PLCγ1 oligonucleotides down-regulate PLD1 and PLCγ1, respectively, compared with α-tubulin controls. (Fii) Monocytes treated with anti-PLD1 oligonucleotides are impaired in their cytosolic calcium signaling after activation of FcγRI by receptor cross-linking using antibody clone 10.1. (Fiii) Monocytes treated with anti-PLCγ1 oligonucleotides are impaired in their cytosolic calcium signaling after activation of FcγRIIa by receptor cross-linking using antibody clone 3D3.

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