Skip to main content
. 2016 Dec 14;3:16034. doi: 10.1038/mto.2016.34

Figure 1.

Figure 1

Phenotypic characterization of ID8-R and CAOV2-R ovarian tumor cells and their parental counterparts. Flow cytometry analysis of CD44 (a) and CXCR4 (b) expression in parental and drug-resistant variants was performed on single-cell suspensions with specific mAbs. Background staining was assessed using isotype control Abs. Data are from one representative experiment of three performed. (c) Susceptibility of ID8-R and CAOV2-R to vaccinia virus infection. The parental and drug-resistant tumor cells were cultured as a monolayer before infection with OVV-EGFP (MOI = 1). The expression of EGFP in infected cells was examined under an immunofluorescence microscope 24 hours later. Scale bars = 25 µm. One representative experiment of three performed is shown. (d) The number of EGFP-expressing cells in each culture was determined by examining single-cell suspensions 24 hours after infection by flow cytometry analysis. Background staining depicts uninfected controls. One representative experiment of four independent experiments performed is shown. (e) Replication of OVV-EGFP in different cultures was determined by titrating viral particles released from the infected cells at different time points by plaque assays in CV-1 cell monolayers. Results are presented as the mean of plaque forming units (PFU)/million cells ± SD of three independent experiments performed in duplicate. *P < 0.05, **P < 0.01, and ***P < 0.001. (f) Phosphorylation levels of Akt and ERK1/2 in tumor cells were determined by Western blotting with antiphospho-Akt(S473-P), antiphospho-Akt(T308-P) and antiphospho-ERK1/2 (Thr202/Tyr204) Abs. Antitotal Akt and antitotal ERK1/2 Abs were used as internal controls and anti-GAPDH Ab was used as a loading control. Bands were developed with HRP-labeled secondary Abs followed by Clarity Western ECL detection system. Representative blot from one experiment out of three performed is shown. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MOI, multiplicity of infection; OVV-EGFP, oncolytic vaccinia virus expressing the enhanced green fluorescence protein.