FIG 5.

Transient G₁/S block and cyclin A1 overexpression are sufficient to enable compatibility of proliferation-dependent H-1PV with cytotoxic GEM. T3M4 cultures were treated with H-1PV at an MOI of 10 PFU/cell, with or without previous exposure to GEM for 12 h at 40 ng/ml, and analyzed by Western blotting and FACS analysis. (A and B) Kinetics of cyclin A1 protein accumulation as monitored by Western blotting sets comprising 4, 10, and 24 hpt and 4, 24, and 48 hpt. The graph depicts the summarized data obtained by quantification of images by use of ImageJ software. (C and D) Cell cycle analyses were performed by PI staining of the DNA in the treated cells and subsequent FACS-assisted measurement of the fluorescence. Analysis of kinetics revealed transient arrest of the cells by GEM at G₁/S and by H-1PV at the S/G₂ boundary by 24 h (bold lines), with consequent release thereafter (48 h) (thin lines). The proportions of the population in the G₀/G₁, S, and G₂/M cell cycle phases were calculated and plotted to show differences from the mock-treated cells.