American Society of Hematology

Figure 1

$Figure 1. PIP2 colocalizes with membrane raft microdomains, the integrity of which is required for lytic granule exocytosis. (A) Primary cultured NK cells were infected with recombinant lentiviruses encoding GFP-PH fusion protein. Live cells were stained with Alexa Fluor 594–conjugated CTxB. GFP-PH and GM1 distribution was analyzed by confocal microscopy (left and middle panels), the colocalization of fluorescence signals is represented in right panel. Representative image from 3 independent experiments is shown. Bar represents 5 μm. (B) Total phospholipids were extracted from whole lysate or from raft and soluble fractions from primary cultured NK cells. Phosphoinositides were resolved by TLC, followed by immunoblotting with anti-PIP2 mAb (top panels). The spot corresponding to PIP2 in raft and soluble fractions was quantified by densitometric analysis; the numbers indicate the percentage of PIP2 distribution between raft and soluble fraction assuming the sum of PIP2 present in both compartments as 100%. One representative experiment of 3 performed is shown. GM1 and β-tubulin immunoblots of the same experiment are shown (bottom panels). (C) Primary cultured NK cells were left untreated or pretreated with MβCD and allowed to interact with a K562-sensitive target. Cell conjugates were fixed, stained with anti-perforin mAb, and analyzed by fluorescence microscopy. The percentage of NK/target cell conjugates containing polarized granules was calculated on randomly acquired fields of 3 independent experiments (means ± SD, n = 100 conjugates). The difference between the treated and untreated group was not significant. (D) Primary cultured NK cells were left untreated or pretreated with the indicated doses of MβCD and allowed to interact with K562 target cells (E:T ratio, 2:1). After 4 hours of stimulation, cells were stained with PE-conjugated anti-CD56 and the percentage of CD107a+ cells was evaluated by cytofluorimetric analysis. Dot-plot analysis of gated CD56+ cells is shown. One representative experiment of 3 performed is shown.$

PIP2 colocalizes with membrane raft microdomains, the integrity of which is required for lytic granule exocytosis. (A) Primary cultured NK cells were infected with recombinant lentiviruses encoding GFP-PH fusion protein. Live cells were stained with Alexa Fluor 594–conjugated CTxB. GFP-PH and GM1 distribution was analyzed by confocal microscopy (left and middle panels), the colocalization of fluorescence signals is represented in right panel. Representative image from 3 independent experiments is shown. Bar represents 5 μm. (B) Total phospholipids were extracted from whole lysate or from raft and soluble fractions from primary cultured NK cells. Phosphoinositides were resolved by TLC, followed by immunoblotting with anti-PIP2 mAb (top panels). The spot corresponding to PIP2 in raft and soluble fractions was quantified by densitometric analysis; the numbers indicate the percentage of PIP2 distribution between raft and soluble fraction assuming the sum of PIP2 present in both compartments as 100%. One representative experiment of 3 performed is shown. GM1 and β-tubulin immunoblots of the same experiment are shown (bottom panels). (C) Primary cultured NK cells were left untreated or pretreated with MβCD and allowed to interact with a K562-sensitive target. Cell conjugates were fixed, stained with anti-perforin mAb, and analyzed by fluorescence microscopy. The percentage of NK/target cell conjugates containing polarized granules was calculated on randomly acquired fields of 3 independent experiments (means ± SD, n = 100 conjugates). The difference between the treated and untreated group was not significant. (D) Primary cultured NK cells were left untreated or pretreated with the indicated doses of MβCD and allowed to interact with K562 target cells (E:T ratio, 2:1). After 4 hours of stimulation, cells were stained with PE-conjugated anti-CD56 and the percentage of CD107a⁺ cells was evaluated by cytofluorimetric analysis. Dot-plot analysis of gated CD56⁺ cells is shown. One representative experiment of 3 performed is shown.

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