Fig 1.
Unc93b expression induces apoptotic cell death.
(a-h) 293T cells were transfected with WT or H412R Unc93b containing a C-terminal Myc tag (a) 48 hours post-transfection, differential interference contrast (DIC) images were obtained from live cells. (b) 48 hours post-transfection, cells were treated with staurosporine for 24 hours and cell death was measured by Cell Titer Glo assay. Data shown is an average of 6 experiments. (c) 72 hours post-transfection, cells were stained with Annexin V and PI and analyzed by flow cytometry. Data shown are representative of 4 experiments. Cell population was determined by gating events on Forward Scatter (FSC) and Side Scatter (SSC). Cells were then gated based on Annexin V and PI staining intensities. (d) 48–72 hours post-transfection, cells were stained with Annexin V and PI and analyzed by flow cytometry as in 1c, and Annexin V+/PI+ populations were compared. Data shown are representative of 4 experiments (e) 293T cells were transfected in parallel with those shown in 1d, lysates were harvested and then subjected to immunoblotting with anti-Myc or -GAPDH antibodies. (f) Prior to transfection (1 hour) cells were treated with zVAD-fmk and then transfected with the indicated constructs for 48 hours (in zVAD-fmk-containing medium). Approximately 48 hours post-transfection, DIC images were obtained from live cells. (g,h) Prior to transfection (1 hour) cells were treated with zVAD-fmk and then transfected with the indicated constructs for 72 hours (in zVAD-fmk-containing medium). 72 hours post-transfection cells were stained with Annexin V and PI and analyzed by flow cytometry as in 1c. Data shown are representative of 2 experiments (g) or average of 2 experiments (h).
Fig 2.
Unc93b is cleaved by caspases during apoptosis.
(a) 293T cells were transfected with the indicated EGFP-fused Unc93b construct and 48 hours post-transfection, cells were treated with TNFα, JSH-23, and Z-VAD-FMK (or mock control) for 18 hours. Lysates were harvested and subjected to immunoblotting with anti-GFP, -PARP, or -GAPDH antibodies. (b) 293T cells were transfected with the indicated EGFP-fused Unc93b constructs and Ha- Flag dual-tagged TRIF and treated with zVAD-fmk 6 hours post-transfection. Lysates were harvested 48 hours post-transfection and subjected to immunoblotting with anti-GFP, -PARP, and–GAPDH antibodies. (c) 293T cells were transfected with the indicated EGFP-fused Unc93b and Flag-tagged TRIF constructs (Full-length, N-terminal or C-terminal); lysates were harvested 48 hours post-transfection and subjected to immunoblotting with anti-GFP and -GAPDH antibodies. In all panels, ns denotes nonspecific bands.
Fig 3.
Unc93b is cleaved by 3Cpro during CVB infection.
(a) 293T cells transfected with EGFP-Unc93b were infected with CVB (MOI = 1), lysates harvested 18 hours post-infection, then subjected to immunoblotting with anti-GFP, -VP1, and -GAPDH antibodies. (b) 293T cells transfected with EGFP-Unc93b were infected with CVB or UV-inactivated CVB (MOI = 1), lysates harvested 18 hours post-infection, then subjected to immunoblotting with anti-GFP, -VP1, and -GAPDH antibodies. (c) 293T cells were cotransfected with the indicated EGFP-fused Unc93b construct and Myc-tagged wild-type (WT) or catalytically inactive C147A 3Cpro, lysates were harvested 48 hours post-transfection and subjected to immunoblotting with anti-GFP and -GAPDH antibodies. (d) 293T cells transfected with the indicated EGFP-fused Unc93b construct were infected with CVB (MOI = 1), lysates were harvested 16 hours post-infection and subjected to immunoblotting with anti-GFP, -VP1, and–GAPDH antibodies. In all panels, ns denotes nonspecific bands.
Fig 4.
Unc93b cleavage does not affect TLR signaling or secretory pathway trafficking.
(a) Schematic of wild-type Unc93b (Unc93bFull), Unc93b-H412R mutant (Unc93bH412R), Unc93b truncation mutants (Unc93bΔ17 or Un93bΔ27) and Unc93b cleavage fragments (Nterm17 and Nterm27). (b, c) 293T-FlagTLR3 (b) or HEK293XL-TLR8HA (c) cells were co-transfected with the indicated Unc93b construct containing a C-terminal Myc tag and/or N-terminal GFP tag, and IFNβ luciferase reporter (pIFNβ-fluc) and control renilla (pRL-null) constructs. Approximately 24 hours post transfection, cells were treated with 1 μg/mL poly(I:C) (b) or 10 μg/mL R848 (c) for 16 hours. Luciferase levels were analyzed using the Dual Luciferase reporter assay system. Firefly luciferase levels were normalized to renilla luciferase levels and then normalized to the vector transfected mock-treated condition. Data shown are an average of 2 independent experiments (b) or a representative experiment (c) and significance is compared to vector plus ligand. (d) Immunoblotting of lysates collected from 293T cells used in panel 4c and probed with antibodies against GFP and Myc (top) or GAPDH (bottom) as a loading control. (e, f) U2OS cells were co-transfected with Venus-fused VSV-G and the indicated Unc93b constructs containing a C-terminal Myc tag. Approximately 48–72 hours post-transfection, cells were fixed and then immunostained for Myc and confocal microscopy performed (e) and then images analyzed to obtain Pearson’s correlation coefficients (f).
Fig 5.
Unc93b cleavage does not affect its induction of cell death.
(a-d) 293T cells were transfected with the indicated Unc93b construct containing a C-terminal Myc tag. (a) 72 hours post-transfection, differential interference contrast (DIC) images were obtained from live cells. (b) 48 hours post-transfection, cell death was measured by Cell Titer Glo assay. Data shown is average of 6 experiments. (c) 72 hours post-transfection, cells were stained with Annexin V and PI and analyzed by flow cytometry. Data was analyzed as described in Fig 1C, gating events on FSC vs. SSC for cell population are not shown here. Data shown are representative of three experiments. (d) 48–72 hours post-transfection, cells were stained with Annexin V and PI and analyzed by flow cytometry. Data were analyzed as described in Fig 1C, and Annexin V+/PI+ populations were compared. Data shown are average of three separate experiments (e) 293T cells were transfected with eGFP-fused Unc93b constructs containing a C-terminal Myc tag. 48–72 hours post-transfection, DIC images were obtained from live cells.