To distinguish between secretion of vesicles and their movement away from the plasma membrane, images were acquired simultaneously in TIRF and epifluorescence.
TIRF and epifluorescence images taken at planes 0, 0.5 and 1.0 ?m above the TIRF plane, before and after secretion. The secreted lytic granule (marked by an arrow) disappeared from the TIRF image and the epifluorescence Oxymatrine
stack images. Scale bar: 3 ?m. Figure S6: The effect on CD3 accumulation can be rescued by expression of a modified Stx7�CTFP construct. A) CTLs were transfected with a combination of control siRNA/modified Stx7�CTFP (upper panel) or Stx7 siRNA/modified Stx7�CTFP (lower panel) and incubated with target cells. After 30 min, cells were fixed and stained with anti-CD3 antibodies. Representative fluorescence images are shown left, corresponding maximum intensity projection (MIP) images are shown right.
CTLs were transfected with a combination of control siRNA/Stx7�CTFP (upper panel) or Stx7 siRNA/Stx7�CTFP (lower panel) and incubated with target cells. Nutlin-3a mw
After 30 min, cells were fixed and stained with anti-CD3 antibodies. Representative fluorescence images are shown left, corresponding MIP images are shown right. Scale bars: 3 ?m. C) Quantification of CD3 fluorescence in proximity to the IS (see Figure 4 for detailed explanation) revealed that the loss of CD3 accumulation can be reversed by expression www.selleckchem.com
of a modified Stx7�CTFP construct, arguing for a specificity of the effect. **p < 0.01. Figure S7: Calcium signaling and accumulation of the pSMAC marker talin are unaffected in Stx7 siRNA-transfected CTLs. A) Representative brightfield and 340/380 nm fura-2 ratio images of CTLs that were transfected with control or Stx7 siRNA. Different colors indicate varying ratios and therefore varying calcium concentrations over time. Different time points show CTLs before and after contact with SEA-pulsed Raji cells. CTLs show an increase in [Ca2+]i only after contact with a pulsed target cell. Cells with no contact showed no increase in [Ca2+]i (not shown). Scale bar: 3 ?m.
Average [Ca2+]i of CTLs transfected with control (n = 335) or Stx7 siRNA (n = 463) from a representative experiment out of two independent experiments. [Ca2+]i signals after contact with pulsed Raji cells are unchanged in Stx7 siRNA-transfected CTLs. C) Representative cells from three independent experiments showing similar accumulation of talin in Stx7 siRNA-transfected cells and control siRNA-transfected cells. The Stx7 siRNA-transfected cells, however, show a defect in CD3 accumulation in contrast to control siRNA-transfected cells. Scale bars: 3 ?m. Figure S8: Stx7 interacts with Stx8, but not with Vti1b, in activated CTLs.