Fig. 4.

TMEM16F-triggered lipid scrambling reduces NK cell cytotoxicity and 2B4 signaling. a Expression of membrane receptors 2B4, SLAMF6, and DNAM-1, as well as adapter SAP, was determined by flow cytometry of YT-S cells expressing empty vector (GFP), wild-type TMEM16F, or the D408G TMEM16F mutant. For detection of SAP, the cells were permeabilized. Open histogram, isotype control antibody; filled histogram, specific antibody. Representative histograms of two independent experiments. b, c YT-S cells expressing GFP alone or in combination with wild-type TMEM16F or D408G TMEM16F were incubated for 6 h with K562 or HeLa cells expressing hCD48 in the presence or in the absence of ionomycin (1 μM) to activate TMEM16F. Cytotoxicity was measured by ⁵¹Cr release at the indicated effector:target (E:T) ratios. The extent of the cell lysis was indicated as the percentage of maximum ⁵¹Cr release. The data are presented as mean ± s.d. of duplicate samples. Representative cytotoxicity assays are shown in (b), while statistics for four independent experiments are depicted in (c). d The indicated YT-S derivatives were preincubated for 5 min at room temperature in the presence or in the absence of ionomycin (10 μM). They were then stimulated or not for 5 min at 37 °C with anti-2B4 antibodies C1.7 and the relevant secondary anti-mouse antibody. Total cell lysates were probed by immunoblotting with anti-phosphotyrosine (p-Tyr; top), anti-phospho-Erk1/2 (pErk1/2; middle) or anti-Erk1/2 (bottom) antibodies. The levels of phospho-Erk1/2 were measured and normalized according to the total Erk1/2 level. Representative blots of three independent experiments. e Calcium influx in the indicated YT-S cells stimulated or not with anti-2B4 antibodies. Calcium influx was determined by the indicated fluorescence ratio for Indo-1. Ionomycin served as the positive control. Representative histograms of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed Student’s t tests). The data are presented as the means ± s.e.m