Figure 7
From: TMEM16F activation by Ca2+ triggers plasma membrane expansion and directs PD-1 trafficking
TMEM16F-null Jurkat T cells internalise PD-1 upon treatment with ionomycin (A) TMEM16F-null Jurkat T cells over-expressing a PD-1/GFP chimera were treated with 5 μM ionomycin for 15 minutes at 37 °C for 15 minutes then chilled and stained at 4 °C for expression of a variety of surface molecules and analyzed by FACS. Data shows the percentage of initial surface expression after ionomycin treatment. (B) TMEM16F-null Jurkat T cells over-expressing a PD-1/GFP chimera were treated with 5 μM ionomycin for 15 minutes at 37 °C then chilled and stained at 4 °C for PD-1 expression. PD-1 staining and cellular GFP expression were analyzed by FACS; data shows the percentage of initial expression after ionomycin treatment. (C) TMEM16F-null Jurkat T cells over-expressing a PD-1/GFP chimera, or a PD-1/GFP chimera where the PD-1 transmembrane sequence was replaced by that from HLA-A2, were treated with 5 μM ionomycin for 15 minutes at 37 °C then chilled and stained at 4 °C for PD-1 expression. PD-1 staining was analyzed by FACS; data shows the percentage of initial expression after ionomycin treatment. (D) TMEM16F-null Jurkat T cells over-expressing a PD-1/mCherry chimera, were imaged by SR-SIM microscopy before and after ionomycin treatment. Arrows indicate fluorescent vesicles (scale bar is 5 μm). Cellular FACS data shows the mean +/− SEM for three independent experiments. (****p < 0.0001, **p < 0.01, Student t-test).
