Supplementary Figure S1. Specific binding of Ad5fk3 to desmoglein 2 (DSG2) and Ad5f35 to CD46. (a) BON cells were incubated with viruses at 1000 evg/cell for 2 hr at 4°C followed by in situ Proximity Ligation Assay (PLA) on fixedcells. Primary antibodies were mouseanti-DSG2 and goat anti-hexon. (b) PLA signals showing proximity between virus and DSG2 receptor were counted in 200 cells from each sample and divided into three groups: 0-10; 10-20; 20-40 PLA signals/cell. (c) BON cells were incubated with viruses at 1000 evg/cell 2 hr at 4°C followed by PLA assay on fixed cells. Primary antibodies were mouse anti-CD46 and goat anti-hexon. (d) PLA signals showing proximity between virus and CD46 receptor were counted in 200 cells from each sample and divided into three groups: 0-10; 10-20; 20-40PLA signals/cell. (a, c)Fluorescence staining of the cells: nuclei - Hoechst (blue); actin - Phalloidin (green); virus-receptor proximity (PLA signal) - Cyanine 3 (red).
Supplementary Figure S2. Transduction efficiency for FWKT-modified Ad5 is inhibited by SSTRs sequestration but not by CAR downregulation. (a)BON cells were incubated with somatostatin-14 (0, 10, 100 nM) for 6 hr at 37°C, followed by transduction with GFP-expressing adenoviral vectors at 50 evg/cell. GFP-positive cells were counted by flow cytometry 24 hr later. Average of two experiments with standard deviation is presented. (b)BON cells were transfected with (10, 30, 90 nM) of CXADR siRNA (mix of four siRNA targeting CAR mRNA) or with 90 nM negative control siRNA (non-related). After 36 hr the cells were transduced with GFP-expressing adenoviral vectors at 50 evg/cell for 2 hr at 37°C. GFP-positive cells were counted by flow cytometry 24 hr later. Average of three experiments with standard deviation is presented. (c) Total protein lysates were prepared 36 hr after transfection with siRNA and 25 µg of samples were resolved by SDS-PAGE. CAR expression levels were detected by Western blotting and β-actin was used as a control of protein loading.
Supplementary Figure S3. CgA gene expression in neuroendocrine tumor samples, normal hepatocytes and cell lines. cDNAs were synthesized using 0.5 g of total RNA from primary carcinoid samples, normal human hepatocytes and a panel of cell lines. Quantitative real-time PCR was performed to detect CgA gene expression and Ct values were calculated from triplicate samples (mean ± SD). Data were evaluated using the 2-ΔΔCT method and expressed in relation to the level of β-actin (set as 1).
Supplementary Figure S4. Production of progeny viruses from infected cells. Foregut (F2livmet) and midgut (M3livmet) carcinoid cells, isolated from liver metastatses, were transduced with oncolytic adenoviruses and AdMock at a concentration of 50 evg/cell, while the neuroendocrine tumor cell lines BON, SH-SY-5Y and SK-N-BE(2) and the melanoma cell line mel526 were transduced at a concentration of 100 evg/cell. After 2 hr of transduction, the cells were washed and incubated at 37°C for 72 hr. Supernatants were collected, diluted (7x) and added to 911 cells for 2 hr. (a) The presence of viral particles in 911 cells was confirmed by FFU assay, 24 hrs after incubation with the diluted supernatants. A primary mouse monoclonal anti-Ad hexon antibody and a secondary Alexa488-labelled goat polyclonal anti-mouse antibody were used to detect viral capsid. (b) Cell viability of 911 cells was examined 48 hr after incubation with the diluted supernatants. Cell viability values were expressed in relation to the viability of untransduced cells. Average enzymatic activities from triplicates samples are shown (mean ± SD).