Figure 1From: Targeting gallbladder cancer: oncolytic virotherapy with myxoma virus is enhanced by rapamycin in vitro and further improved by hyaluronan in vivoMyxoma virus productively infects human gallbladder cancer cell lines in vitro and phosphorylated Akt expression levels in human gallbladder cancer cell lines. A. Replication over a period of 1 replication cycle was investigated using high multiplicity of infection (MOI) single-step growth curves in control permissive CV-1, GBC cell lines (GBC-SD, NOZ, SGC-996). All cells were infected with vMyx-gfp (MOI = 5), and cell lysates were collected at the indicated time points after infection. Viral titers were determined by titration in CV-1 cells. B. Replication over a period of multiple replication cycles was investigated using low MOI multi-step growth curves. CV-1, GBC-SD, NOZ and SGC-996 were infected with vMyx-gfp (MOI = 0.01). C. GFP was in visualized by fluorescence microscopy. GBC-SD, NOZ, SGC-996, a permissive glioma cell line control (U251), and a poorly-permissive murine fibroblast cell line control (NIH3T3) were infected with vMyx-gfp at an MOI = 5 and photographed 48 h after infection. D. Effects of MYXV on cell viability of GBC cell lines in vitro. E. Early viral protein was determined by M-T7 expression, and late viral protein was determined by Serp-1 expression at the indicated time points by western blot of cell lysates. F. The expression of phosphorylated Akt (Thr308) in U251, GBC-SD, NOZ, SGC-996, and NIH3T3 cells was evaluated by western blotting. G. Densitometry results of relative p-Akt expression normalized by β-Actin of each cell line in Figure 1F. H. GBC-SD, SGC-996, and NOZ were pretreated with Rap (20 nmol/L or 100 nmol/L) for 1 h, and then cells were infected with vMyx-gfp (MOI = 5). Levels of p-Akt (Thr308) and total Akt in cell lysates were determined by western blotting. I. Densitometry results of relative p-Akt expression normalized by β-Actin of each cell line in Figure 1H. FFU, fluorescent focus-forming units.Back to article page