FAST protein expression in A549 cells decreases cellular metabolic activity, promotes apoptosis and concomitant loss of membrane integrity.

<p><b>A)</b> A549 cells were infected at an MOI of 10 with AdEmpty or AdFAST. Cellular metabolic activity was assessed every 24 h until 96 hpi using an MTS assay. The average of three independent experiments (n = 3) done in triplicate is plotted with the standard error of the mean. Values were normalized to mock infected cells at 24 hpi. *p<0.05 compared to mock infected cells at their corresponding timepoints. **p<0.05 comparing AdFAST to AdEmpty infected cells. <b>B)</b> A549 cells were infected with AdEmpty or AdFAST at an MOI of 1, 10, 50 or 100. An MTS assay was conducted 72 hpi. The average of three independent experiments (n = 3) done in triplicate ± the standard error of the mean is shown with normalization to mock infected cells. *p<0.05 compared to mock infected cells. **p<0.05 comparing AdFAST to AdEmpty treated cells. <b>C)</b> A549 cells were treated with AdEmpty or AdFAST at an MOI of 10, or left uninfected, and crude protein extracts prepared 24 hr later and examined for total or cleaved caspase 3 by immunoblot. As positive controls, A549 cells were treated with 100 μM etoposide for 24 h, or 1 μM staurosporine for 6 h. Alpha-tubulin served as a loading control. <b>D)</b> A549 cells were infected with AdEmpty, AdFAST or VSVΔ51 using a range of MOI. Relative cell membrane integrity was measured based on the lactate dehydrogenase levels in the supernatant 72 hpi. The average of two independent experiments (n = 2) is shown with each experiment done in triplicate. Error bars denote the standard error of the mean. *p<0.05 compared to cells only. **p<0.05 when comparing AdFAST to AdEmpty infected cells.</p

FAST protein expression induces extensive cell fusion in 293 cells.

<p><b>A)</b> 293 cells were infected with AdEmpty or AdFAST at an MOI of 1 and stained with Giemsa stain 18 h later. Images were captured using bright field microscopy (20x objective). A region of fused cells for the AdFAST-treated cells is outlined with a dotted line, and several nuclei within the syncytium are indicated with asterisks (*) <b>B)</b> Fusion index of 293 cells infected with AdEmpty or AdFAST. The fusion index for two fields of view were determined, and the average with the standard deviation is depicted in the graph. <b>C)</b> 293 cells were infected with AdRFP or AdFAST/RFP at MOI 10 and observed using fluorescence microscopy at 48 h post infection. All images were taken using a 20x objective. <b>D)</b> 293 cells were infected at a MOI of 10 with AdEmpty or AdFAST and relative metabolic activity was determined using an MTS assay over a 96 h interval every 24 h. Experiments were completed in triplicate and the average of three independent experiments is shown (n = 3). Values were normalized to mock infected cells at 24 hpi. Error bars denote the standard error of the mean. *p<0.05 compared to mock infected cells. **p<0.05 when comparing AdFAST to AdEmpty infected cells.</p

Ad-mediated FAST protein expression from an E1-deleted vector does not affect virus growth or yield.

<p><b>A)</b> All viruses are early region 1 (E1) and early region 3 (E3) deleted. AdFAST-HA has a single HA tag linked to the C terminus through a glycine linker. ITR denotes inverted terminal repeats and Ψ is the packaging signal. RFP is the red fluorescent protein and CMV represents the cytomegalovirus enhancer/promoter. <b>B)</b> 293 and A549 cells were infected with the control virus AdEmpty or AdFAST-HA at an MOI of 10. Whole cell lysates were collected 48 hpi. Samples were probed for the HA tag, Ad5 fibre and alpha-tubulin for a loading control. The top band denoted by * in the A549 samples is non-specific. <b>C)</b> 293 cells were infected with AdEmpty or AdFAST at an MOI of 1 and whole cell lysates were collected at 6, 18, 24 and 30 hpi. Samples were probed for Ad5 fibre and alpha-tubulin was used as a loading control. <b>D)</b> Supernatants collected from 293 cells infected with AdEmpty or AdFAST at an MOI of 1 were used to conduct plaque forming assays to determine the number of viral progeny at various times post-infection.</p

AdFAST does not induce fusion or promote survival in immunodeficient CD-1 mice with subcutaneous A549 tumors.

<p><b>A)</b> CD-1 nude mice harbouring subcutaneous A549 tumors were intratumorally injected with PBS, 5x10⁸ pfu AdEmpty or AdFAST. Five days post injection, tumors were excised, fixed, sectioned and subjected to hematoxylin and eosin staining. Results are representative of 2–3 mice (10x objective). <b>B)</b> CD-1 mice with subcutaneous A549 tumors intratumorally injected with PBS, 5x10⁸ pfu AdEmpty or AdFAST were measured at day 20 post injection. The line in each column represents the average of the associated treatment group. <b>C)</b> A Kaplan-Meier survival curve shows the percentage survival of CD-1 mice with subcutaneous A549 tumors intratumorally injected with PBS, AdEmpty or AdFAST over time. Each treatment group consisted of 5 mice.</p