Legends to supplemented information
Figure 1-sup:Amino acid sequence comparison of NDV-HUJ and two other attenuated NDV strains, Hitchner B1 and LaSota.
The entire nucleotide sequence of NDV-HUJ genome was translated and aligned against sequences of NDV Hitchner B1and NDV LaSota, using ClustalW. The bold letter amino acids represent differences in the sequence. The amino-acid number represents the position in the noted gene.
Figure 2-sup: NDV-HUJ is oncolytic to chemo-resistant primary melanoma cultures
(A-D): Melanoma primary cultures were mock or NDV-HUJ infected (MOI=10). Parallel cultures were treated with a panel of chemotherapeutic drugs:0.5μM Cisplatin, 10μM Carboplatin,1μM Oxaplatin or30μM Etoposide. The concentration of chemotherapeutic drugs used was previously applied to induce cell death in primary melanoma cells (16). Cells were harvested 48 hrs post treatment and taken to FACS analysis for DNA content. The Y axis represents the fraction of cells in the sub-G1 phase of cell cycleindicating apoptotic cells. Numbers in parenthesis above the bars represent fold increase in cell death due to NDV-HUJ infection relative to mock control cells.
Figure 3-sup: Livin siRNA rescuesADVmelanoma fromthe oncolytic effect of NDV-HUJ
Melanoma primary culture 351 ADV wastransduced with GIPZ™ lentiviral vector expressing GFP and siRNA against Livin or a control siRNA (see Materials and Methods). Cultures express GFP (Y axis represent GFP positive cells) 24hrs post lentiviral transduction. Transduced cultures were either mock or NDV-HUJ (MOI=10) infected. Cultures were harvested after 48 hrs (upper panel) and 72 hrs (lower panel) post infection and stained with PI to determine cell death by FACS analysis. Cells were first selected for GFP expression (Lenti vector transduced cells represent about 90% of total cells marked as G+) and PI staining was determined on this cell fraction. The results presented are based on doubly stained cells that are positive for both GFP and PI. The results demonstrate that the amount of dead cells (PI+/GFP+) in the NDV-HUJ infected Livin siRNA sample is significantly lower than that of NDV-HUJ infected control siRNA cells 48 as well as 72 hrs post infection.
Figure 4-sup:Effect of Livin knockdown on viral protein synthesis
NDV-HUJ protein synthesis was monitored by FACS analysis for viral protein antigens. 351 ADV melanoma cells expressing either "Livin siRNA" (Gray) or "control siRNA" (Black) were either mock (filled histogram) or NDV-HUJ (MOI=10) infected. Cells were harvested 1hr (dash line) and 24 hrs (solid line) post infection and stained with anti NDV antibodies (anti NDV chicken serum + donkey anti chicken IgG CY5- conjugated). Cells were quantifiedto viral surface proteins amountusing the Fluoresce Geometric Mean (F.G.M) value as described in Materials and Methods. The fluoresce intensity correlates with the amount of viral protein(25).
Figure 5-sup: Induction of IFN-beta gene is functional in the melanoma primary cultures
RNA was prepared from mock or NDV-HUJ (MOI =10) infected primary melanoma cultures 24 hrs post infection. RNA (1ug) was subjected to RT-PCR amplification to trace the IFN-beta mRNA using the following primers: IFN-β forward (5'-ACATCCCTGAGGAGATTAAGCA-3') and IFN-β reverse (5'-GCCAGGAGGTTCTCAACAATAG-3'). The IFN primers produced a DNA of 150 base pairs. DNA products were collected after 25, 30 and 35 PCR cycles. Results presented are from the linear range of the amplification, 30 cycles. The RT-PCR reactions were always carried out in the presence and absence of RT to ensure detection of RNA only (data not shown). Relative IFN-beta mRNA was calculated as described in Materials and Methods and (25).
Figure 6-sup: Secretion of biologically active IFN from NDV-HUJ infected primary melanoma cultures.
In order to analyze biological active IFN induction in the melanoma cells by NDV-HUJ, media from mock and NDV-HUJ (MOI=10)infected 162 EAR and 351 ADV melanoma cultures were taken48 hrs post infection. The conditioned media was centrifuged for 30 minuets at 10000xg. Clear media (diluted 1:4, 1:12, 1:20) was added to 351 ADV melanoma cultures 16 hrs before NDV-MTH (6) infection (MOI=0.2). As a control, anti NDV chicken serum (1:500) was added to infected culture 1 hr post infection. Cells were harvested 24 hrs post infection and double stained with anti NDV antibodiesand PI. Samples were analyzed by flow cytometry as described in Materials and Methods. NDV-MTH is a pathogenic (mesogenic) strain that replicates and spread in mammalian cells (6).
The results indicated that both infected EAR and ADV melanoma cultures produce biologically active IFN that protects from cell death and inhibits NDV protein synthesis when added to cultures 24 hrs before the infection. The level of IFN produced in the infected ADV melanoma culture appears higher than that in the EAR cultures. This is further evidence that the selective sensitivity of ADV melanomas to the oncolytic activity of NDV is due to expression of Livin rather that production of IFN.