1. Volume 15, Issue 8, Pages 1531-1536 (August 2007)
The RAS/Raf1/MEK/ERK Signaling Pathway Facilitates VSV-mediated Oncolysis: Implication for the Defective Interferon Response in Cancer Cells 
Josh A Noser, Amber A Mael, Ryuta Sakuma, Seiga Ohmine, Paola Marcato, Patrick WK Lee, Yasuhiro Ikeda 
Molecular Therapy 
Volume 15, Issue 8, Pages (August 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Involvement of RAS effector pathways in vesicular stomatitis virus (VSV)-mediated oncolysis. (a) 3T3 cells were infected with retroviral vectors coding for the active form of RAS (RAS12V) and two RAS mutants (12V35S and 12V40C), and an empty vector control (Ctrl). Western blotting was performed to verify RAS expression in the 3T3 cell lines. (b) The cells were infected with VSV expressing green fluorescent protein (GFP) at a multiplicity of infection (MOI) 0.05 and the cytopathic effects were observed 18 hours after infection. Following VSV infection, efficient oncolysis was evident in the RAS12V35S (non-RalGEF or PI3 kinase signaling)-expressing but not in the RASV12C40 (non-Raf/MEK/ERK signaling)-expressing cell lines. (c) Detection of elevated levels of phosphorylated ERK (p-ERK1 and p-ERK2) in the 3T3 cells over-expressing the active-form of Raf1 by Western blotting. (d) Control, active-forms of RAS- or Raf1-expressing 3T3 cells were infected with the GFP-expressing VSV at an MOI of Twelve hours after infection, GFP-positive cells were analyzed by fluorescence-activated cell sorting. The numbers indicate the percentage of GFP-positive cells. The mean fluorescent intensity (MFI) of the GFP-positive population is also shown. UV, ultraviolet; TM, transmission.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Involvement of the RAS/Raf1/MEK/ERK signaling pathway in the defects of interferon α(IFNα) system. (a) 3T3 RAS, Raf1, and empty vector control cells were tested for responsiveness to INFα and/or U0126 treatment. A p38 inhibitor, SB203580, was used as a control. Expression of RAS 12V or Raf1 rendered cells less responsive to IFNα treatment. (b) Progeny viral production from the IFNα and/or U0126-treated cells. The IFNα- or U0126-treated cells were infected at a multiplicity of infection of 0.05 for 24 hours. The culture supernatants were harvested and their viral titers were measured on 293T cells as described in Materials and Methods. (c) Negative regulation of the IFNα-mediated antiviral response by RAS in primary human cardiac fibroblast cells. Western blot analysis was performed to ensure RAS expression. The cells were infected as before and the viral titers were measured at 24 hours after infection on 293T cells.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Restoration by U0126 of defects in the interferon α (IFNα) response of cancer cells. (a) Cancer-derived cell lines were pre-treated with IFNα and U0126 for 12 hours then infected at a multiplicity of infection (MOI) of Two days after infection, the infected cells were observed under the microscope. (b) Culture supernatants were harvested 18 hours after infection at an MOI The viral titers were determined on 293T cells.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Dual treatment with interferon α (IFNα) and U0126 reduced the levels of viral RNAs in cancer cells. (a) In order to examine the effects of IFNα and/or U0126 treatment on viral protein synthesis, Western blotting was performed to detect vesicular stomatitis virus (VSV)-specific protein expression in the infected cells. (b) VSV-specific negative- and positive-stranded RNAs were detected by reverse transcriptase polymerase chain reaction (RT-PCR). As a control, α-tubulin complementary DNA (cDNA) was amplified by RT-PCR. (c) Primary human cardiac fibroblast (HCF) cells and cancer cell lines were treated with IFNα and/or U0126 overnight. Induction of MxA-specific RNA was examined by RT-PCR. As a control, the same RNA samples were utilized to amplify α-tubulin cDNA.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions