Expression of RavK causes cytotoxicity in both yeast and mammalian cells and reduces the F-actin content in mammalian cells.
A. A schematic diagram of RavK. The blue box highlighted the position and the sequence of the predicted HEXXH motif. B. Expression of RavK induces yeast growth arrest in an H95EXXH99-dependent manner. Yeast strains expressing RavK or the indicated mutants under the control of the galactose-inducible promoter were serial-diluted and spotted onto plates containing glucose or galactose, respectively. Plates were incubated at 30°C for 48 h before image acquisition. C. Expression of RavK and the indicated mutants in yeast. Yeast strains grown in glucose medium to saturation were washed with water 5 times and split equally to 2 halves. One half was frozen immediately (sample 1), the other half was induced in galactose medium for 8 h (sample 2). Total proteins of all samples were resolved by SDS/PAGE and probed by immunoblotting with a RavK-specific antibody. The 3-phosphoglycerate kinase (PGK) was used as a loading control. D. RavK reduces F-actin content in COS-1 cells. COS-1 cells transfected by the indicated plasmids for 24 h were fixed and subjected to Texas-red-conjugated phalloidin staining. Images from one representative were shown and similar results were obtained in at least three experiments. Bar, 20 μm. E. Integrated pixel density of phalloidin staining in cells expressing indicated proteins plotted as average F-actin intensity per cell. N>60 per condition; error bars represent standard error of the mean (SEM); A.U., arbitrary units; ****, p<0.0001. F. The spread cell area of cells expressing indicated proteins plotted as average area per cell. N>60 per condition; N.S., not significant; *, p<0.05.