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. Author manuscript; available in PMC: 2019 Feb 1.
Published in final edited form as: J Immunol. 2017 Dec 20;200(3):1110–1123. doi: 10.4049/jimmunol.1701113

Figure 7. TRAILshort protection from TRAIL can be transferred to neighboring cells.

Figure 7

(A) Effector 293T cells expressing either TRAILFL, TRAILshort, or both were mixed with CTO-labeled target Jurkat T cells. Following incubation cells were analyzed by flow cytometry, gating on CTO positive (Jurkat) populations and staining for Live/Dead cells (Green). (B) 293T cells were transfected with 1, 2, 5, 10, or 20 μg of N-terminal HA-tagged TRAILFL alone and analyzed by Western with an anti-HA antibody. (C) 293T cells were co transfected with 1, 2, 5, or 10 μg of TRAILshort and a constant 10 μg TRAILFL and analyzed by Western blot. (D) 293T effector cells transfected with increasing amounts of TRAILFL from (B) were co-cultured with CTO+ target Jurkat cells and the percentage CTO+ Jurkat cell killing was determined. (E) 293T effector cells from (D), co-transfected with increasing amounts of TRAILshort at constant TRAILFL, were co-cultured with target cell and analyzed as in (A). D, E represent mean (SD) of four independent experiments. (F) Schematic of plasmids encoding N-terminal Ruby-tagged-TRAILshort (Ruby-TRAILshort), a construct missing the transmembrane domain (Ruby-TRAILshortΔTM), and the Ruby tag alone (bottom panel). Western analysis with anti-TRAILshort antibody and anti-actin controls of lysates from 293T cells transfected with these constructs (top panel). (G) 293T cells were transfected with the constructs in (F) and representative micrographs are shown (top panel). Supernatants from the 293T transfected cells were harvested and used to treat target HeLa cells which were then analyzed by confocal microscopy. Representative micrographs superimposing the blue (DAPI stained HeLa nuclei) and red (ruby, ruby-TRAILshort or Ruby-TRAILshortΔTM) filter channels are shown (bottom panel). (H) HeLa cells from (G) that were pre-treated with supernatants from 293T transfected with Ruby-TRAILshort or Ruby-TRAILshortΔTM were treated with sk-TRAIL. Cell killing was quantified by active caspase 3 staining (Green) over time. Data representative of four independent experiments. P<0.05 considered statistically significant by linear regression.