Table 3.

Neither ATP nor tubulin- or actin-mediated cytoskeletal movement is required for HLA-C clustering by KIR

Compound added	Concentration	Time of incubation, hr	n, number of conjugates imaged	% clustering	% ATP depletion
d-glucose	50 mM	2	153	72	0
2-Deoxyglucose	50 mM	2	182	72	89
Azide	50 mM	2	137	79	94
Antimycin-A	13 μM	1	175	66	97
2,4-Dinitrophenol	450 μM	1	88	75	44
Rotenone	25 μM	1	111	68	43
Colchicine	10 μM	1	42	78	N/A
Cytochalasin B	10 μM	1	83	75	N/A
Cytochalasin D	10 μM	1	183	66	N/A

Table shows the number of 221/Cw6-GFP cells with a cluster of GFP fluorescence at the contact with YTS/KIR1 in the presence of metabolic or cytoskeletal inhibitors. The extent of ATP depletion is shown for each metabolic inhibitor. By calibrating the luciferase assay with known ATP concentrations, the total amount of ATP in 50 × 10³ of untreated NK and target cells (25 × 10³ of each) was about 2 × 10⁻¹⁰ moles. The time of incubation indicated refers to the time that each cells type was preincubated before the cells were mixed together. Cells then were incubated together for a further 20 min in the presence of the compounds. “% clustering” refers to the number of target cells that have a cluster of HLA-C at an NK cell contact. These data do not preclude the possibility that the number of cell conjugates formed or the quantity of clustered protein is affected by the compounds used. N/A, not applicable.