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Supplementary Figure Legends:
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Figure S1: Regulated degranulation elicited by PMA and ionomycin in NKL cells.
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(A) NKL cells on poly-L-lysine coated glass coverslips were stimulated with PMA and
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ionomycin and imaged by TIRF microscopy in the presence of 0.016 µM Alexa Fluor
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647-conjugated CD107a F(ab). A stimulated NKL cell (upper panel) and an unstimulated
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NKL cell (bottom panel) were imaged by TIRF microscopy. Movie frames from the
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indicated time points are shown. Corresponding DIC images are shown on the left. The
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time point ‘0’ indicates the start time point at which TIRF images were acquired. Note
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the dynamic and dispersed distribution of exocytosed LAMP-1 (upper panel). Very little
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LAMP-1 is detected in the absence of PMA and ionomycin treatment (bottom panel).
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Note that 0.016 µM Alexa Fluor 647-conjugated CD107a F(ab) did not label NK cells
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non-specifically. The scale bars are 5 µm. (B) Maximum fluorescence intensity (FI) for
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individual NKL cells treated either with DMSO vehicle (Control) or with PMA and
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ionomycin. The average and standard deviation of the mean are indicated. Statistical
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significance was determined using the t-test. Data are representative of two independent
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experiments.
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Figure S2: Complete fusion of a granzyme B-DsRed containing lytic granule in live
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NKL cell under TIRF microscopy.
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Time-lapsed images taken at ~ 20 min from a fusion event after addition of PMA and
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ionomycin. Images were acquired by TIRF microscopy. The scale bar is 500 nm. The
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images are representative of at least 5 fusion events from 12 cells in two independent
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experiments.
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Figure S3: Colocalization of granzyme B-DsRed with GFP-FasL in live NKL cells.
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NKL cells were double transfected with granzyme B-DsRed and GFP-FasL. Images were
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acquired by Meta-510 Zeiss confocal microscopy. The scale bar is 5.0 µm. The images
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are representative of at least 50 cells in two independent experiments.
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Figure S4: Colocalization of GFP-FasL with perforin containing LG in NKL cells.
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Stably transfected NKL-GFP-FasL cells were fixed and permeabilized on Poly-L-Lysine
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coated coverslips. Perforin containing lytic granules were stained by anti-perforin
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monoclonal antibody followed by Alexa Fluor conjugated secondary antibody. The scale
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bar is 3.0 µm. The images are representative of 10 cells in two independent experiments.
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Figure S5: Complete fusion of two DsRed-FasL-pHluorin positive lytic granules in
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live NKL cell under TIRF microscopy.
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Time-lapsed images taken at ~ 38 min from two complete fusion events after addition of
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PMA and ionomycin. Sequential TIRF microscopy images of single LG in complete
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fusion event #1 and #2 are shown. Images were acquired by TIRF microscopy. The scale
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bars are 1.0 µm. The images are representative of at least 5 fusion events from 20 cells in
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two independent experiments. The weak DsRed signal in the stably transfected NKL cells
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may be due to proteolytic cleavage in the cytoplasmic tail of DsRed-FasL-pHluorin.
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Figure S6: DsRed-FasL-pHluorin positive lytic granules in primary NK cells do not
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undergo spontaneous fusion.
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Primary, unstimulated NK cells were transiently transfected with DsRed-FasL-pHluorin
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and attached onto poly-L-lysine coated coverslips. Dual color images were acquired
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simultaneously by TIRF microscopy equipped with GFP/DsRed dual-view microimager.
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The scale bar is 500 nm. The images are representative of 65 lytic granules from 13 cells
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in two independent experiments. Note that a DsRed signal was observed but without a
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pHluorin fluorescence signal.
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Supplementary Movie Legends
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Movie S1: Live images of regulated degranulation elicited by PMA and ionomycin.
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Accumulation of Alexa Fluor 647-CD107a Fab over time under PMA and ionomycin
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stimulation (upper-left). In the upper-right panel, the updated mean fluorescence
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intensity of CD107a over time on the highlighted region by a purple square is shown over
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time. Note that the time interval between frames is 5 seconds. The scale bar is 5 µm. The
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images were converted into gray scale 8 in Image-pro-plus software. In contrast, very
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little CD107a staining was observed in NKL cells in the absence of PMA and ionomycin
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(bottom panel). The time point ‘0’ indicates the start time point at which TIRF images
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were acquired. The data is representative of NK cells that have been undergoing
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degranulation.
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Movie S2: Complete fusion of LG visualized with GFP-FasL.
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Single color TIRF imaging was carried out in NKL cell expressing GFP-FasL. The arrow
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indicates the LG that is undergoing complete fusion under TIRF microscopy. LG
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approached the PM, appeared to dock, and rapidly fused with the PM, releasing a bright
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fluorescence cloud that diffused at the PM, consistent with complete fusion. Scale bar is 3
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µm.
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Movie S3: Visualization of incomplete fusion by double labeling of LG membrane and
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cargo protein.
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Two-color TIRF imaging was carried out in NKL cell co-expressing granzyme B-DsRed
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(LG cargo protein) and GFP-FasL (LG membrane protein). This movie was acquired by
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simultaneous illumination of pHluorin and DsRed using a GFP/DsRed dual-view
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microimager. The arrow indicates the LG that is undergoing incomplete fusion. Scale bar
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is 3 µm.
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Movie S4: Addition of ammonium chloride (NH4Cl) to raise the pH in LG induced
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strong pHluorin fluorescence.
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Two-color confocal imaging was carried out in NKL cell expressing DsRed-FasL-
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pHluorin after addition of 500 mM NH4Cl (pH 7.4) solution. Imaging was acquired at 20
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seconds per frame. The movie was played at 5 frames per second. Scale bar is 10 µm.
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Please note the cell labeled with circle. The pHluorin fluorescent signals (Green)
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increased as the DsRed fluorescent signals (Red) are relatively constant.
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Movie S5: Visualization of incomplete fusion event using DsRed-FasL-pHluorin probe.
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Two-color TIRF imaging was carried out in NKL cell expressing DsRed-FasL-pHluorin.
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The DsRed and pHluorin fluorescent signals were acquired simultaneously by dual-view
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microimager under TIRF microscopy. The left panel is pHluorin signal. The right panel is
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DsRed signal. The circle indicates the LG that is undergoing incomplete fusion. Scale bar
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is 0.5 µm.
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Movie S6: Visualization of complete fusion event using DsRed-FasL-pHluorin probe.
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Two-color TIRF imaging was carried out in NKL cell expressing DsRed-FasL-pHluorin.
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The DsRed and pHluorin fluorescent signals were acquired simultaneously by dual-view
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microimager under TIRF microscopy. The left panel is pHluorin signal. The right panel is
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DsRed signal. The arrow indicates the LG that is undergoing complete fusion. Scale bar
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is 3.0 µm.
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